Method for producing and purifying RNA, comprising at least one step of tangential flow filtration

ABSTRACT

The present invention relates to method for producing and purifying RNA comprising the steps of providing DNA encoding the RNA; transcription of the DNA into RNA; and conditioning and/or purifying of the solution comprising transcribed RNA by one or more steps of tangential flow filtration (TFF).

This application is a continuation of U.S. application Ser. No.16/934,279, filed Jul. 21, 2020, which is a continuation of U.S.application Ser. No. 15/580,092, filed Dec. 6, 2017, now U.S. Pat. No.10,760,070, which is a national phase application under 35 U.S.C. § 371of International Application No. PCT/EP2016/062152, filed May 30, 2016,which claims benefit of International Application No. PCT/EP2015/062002,filed May 29, 2015, the entire contents of each of which are herebyincorporated by reference.

This invention was made with government support under HR0011-11-3-0001awarded by the Defense Advanced Research Projects Agency. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The present invention relates to methods of tangential flow filtration(TFF) for producing and purifying RNA.

BACKGROUND OF THE INVENTION

RNA is emerging as an innovative candidate for a variety ofpharmaceutical applications, but efficient purification is continuing tobe a challenge. This is partly due to the different types andcombinations of undesired contaminants in a sample that need to beseparated from a desired RNA species to obtain a pure RNA sample. Suchcontaminants are typically components and by-products of any upstreamprocesses, for example RNA manufacture. Where in vitro transcription isused to manufacture large RNA, following successful transcription thesample typically contains the desired RNA species alongside variouscontaminants such as undesired RNA species, proteins, spermidine, DNA orfragments thereof, pyrophosphates, free nucleotides, endotoxins,detergents, and organic solvents.

Commercial downstream applications (e.g. formulation and use as apharmaceutical composition and/or vaccine) pose further constrains onany purification method for RNA requiring (i) a high degree of puritywhile retaining RNA stability and functionality; (ii) compatibility withany formulation requirements of the RNA for in vivo delivery; and (iii)compliance with good manufacturing practices. Furthermore, in order tofacilitate industrial applications, any RNA purification method mustenable consistent, cost- and time-efficient operation (e.g. quick, easy,reproducible, high yield purification on a large scale).

RNA precipitation allows sample concentration as well as depletion ofcontaminating high molecular weight contaminants and low molecularweight contaminants (e.g. proteins and spermidine, respectively).However, precipitation is not the method of choice in large-scaleproduction processes since precipitation and resolubilization of nucleicacids is time consuming. Moreover, the use of alcohols and other organicsolvents should be avoided in large-scale (good) manufacturingprocesses.

Methods for the purification of RNA are known in the art. Pascolo et al.(Methods Mol Med 2006; 127:23-40) describes a method for thepurification of mRNA from an in vitro transcription reaction sample inanalytical scale (purification of 25 μg RNA in 20 μl sample volume). Themethod involves DNase treatment followed by precipitation of the longermRNA with lithium chloride. However, the authors report that this methoddoes not provide RNA of high purity, as it does not completely removecontaminants such as DNA and protein. Furthermore, the method involvesthe use of organic solvents and is laborious and time-consuming,involving as many as 36 steps requiring extensive manual sample handlingat different conditions, including at least one overnight incubationstep. Therefore, while this procedure may satisfy requirements forresearch and laboratory-scale RNA purification, it suffers from a lowdegree of RNA purity, reproducibility and is unsuitable for purificationof pharmaceutical-grade RNA on a commercial scale for implementation inan industrial process.

WO2008/077592 discloses a method for purifying RNA on a preparativescale with ion-pairing reverse phase HPLC using a porous reversedstationary phase. It is reported that a particular advantage of usingthe specified porous stationary phase is that excessively high pressurescan be avoided, facilitating a preparative purification of RNA.

WO2014/140211 discloses a method for purifying large RNA from a sample,comprising steps of tangential flow filtration, hydroxyapatitechromatography, core bead flow-through chromatography or anycombinations thereof. It is also disclosed that it is preferred that nosalts, other than buffering salts, are added to the buffer for thetangential flow filtration. The tangential flow filtration is performedusing a hollow fibre membrane. However, the described nucleic acidloading amounts of the membrane are very low and would therefore requirehuge membrane areas for the large-scale production (g to kg) of mRNA.

WO2014/152966 discloses a method for purifying in vitro transcribed RNA,wherein after RNA in vitro transcription the reaction mixture is treatedwith a protein denaturing agent such as urea and then subjected totangential flow filtration using a hollow fiber membrane.

There remains a need for further RNA purification methods, and inparticular for those that allow cost- and time-efficient purification ofRNAs at an industrial scale with high yield and pharmaceutical-gradepurity, stability and/or shelf life.

It is thus an object of the present invention to provide further RNAproduction and purification methods suitable for large scale RNApreparation.

SUMMARY

The objective is solved by a method for producing and purifying RNA,comprising the steps of

A) providing DNA encoding the RNA;

B) transcription of the DNA to yield a solution comprising transcribedRNA; and

C) conditioning and/or purifying of the solution comprising transcribedRNA by one or more steps of tangential flow filtration (TFF).

Definitions

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

Enzyme: Enzymes are catalytically active biomolecules that performbiochemical reactions such as DNA dependent RNA transcription (e.g. RNApolymerases), or double stranded DNA digestion (e.g. restrictionendonucleases). Enzymes are typically composed of amino acids and/or RNA(ribozymes, snRNA).

Restriction endonucleases: Restriction endonucleases or restrictionenzymes are a class of enzymes that occur naturally in bacteria and insome viruses. Restriction endonucleases can be used in the laboratory tocleave DNA molecules into smaller fragments for molecular cloning andgene characterization. Restriction enzymes bind specifically to andcleave double-stranded DNA at specific sites within or adjacent to aparticular sequence known as the recognition site. Most of therestriction enzymes recognize a specific sequence of nucleotides thatare four, five or six nucleotides in length and display twofoldsymmetry. Some cleave both strands exactly at the axis of symmetry,generating fragments of DNA that carry blunt ends; others cleave eachstrand at similar locations on opposite sides of the axis of symmetry,creating fragments of DNA that carry single-stranded termini (cohesiveends). The restriction endonucleases are categorized into four groups(Types I, II, III, and IV) based on their composition and enzymecofactor requirements, the nature of their target sequence, and theposition of their DNA cleavage site relative to the target sequence. Alltypes of enzymes recognize specific short DNA sequences and carry outthe cleavage of DNA, yielding specific fragments with terminal5′-phosphates.

Restriction endonucleases recognize and bind particular sequences ofnucleotides (the ‘recognition site’) on DNA molecules. Once bound, theycleave the molecule within (e.g., BamHI), to one side of (e.g., SapI),or to both sides (e.g., TspRI) of the recognition sequence. Particularlypreferred is the use of the following restriction enzymes: BciVI (BfuI),BcuI (SpeI), EcoRI, AatII, AgeI (BshTI), ApaI, BamHI, BglII, BlpI(Bpu1102I), BsrGI (Bsp1407), ClaI (Bsu15I), EcoRI, EcoRV (Eco32I),HindIII, KpnI, MluI, NcoI, NdeI, NheI, NotI, NsiI, Mph1103I), PstI,PvuI, PvuII, SacI, SalI, ScaI, SpeI, XbaI, XhoI, SacII (Cfr42I), XbaI.Restriction enzymes recognize short DNA sequences and cleavedouble-stranded DNA at specific sites within or adjacent to thesesequences. Approximately 3,000 restriction enzymes, recognizing over 230different DNA sequences, have been discovered. They have been foundmostly in bacteria, but have also been isolated from viruses, archaeaand eukaryotes. A list of known restriction enzymes can be found at therebase database: http://rebase.neb.com/rebase/rebase.html

Restriction site: A restriction site, also called restriction enzymerecognition site, is a nucleotide sequence recognized by a restrictionenzyme. A restriction site is typically a short, preferably palindromicnucleotide sequence, e.g. a sequence comprising 4 to 8 nucleotides. Arestriction site is preferably specifically recognized by a restrictionenzyme. The restriction enzyme typically cleaves a nucleotide sequencecomprising a restriction site at this site. In a double-strandednucleotide sequence, such as a double-stranded DNA sequence, therestriction enzyme typically cuts both strands of the nucleotidesequence. Most restriction endonucleases recognize palindromic orpartially palindromic sites. A palindrome is defined as dyad symmetryaround an axis. For example, EcoRI digestion produces “sticky” ends,whereas SmaI restriction enzyme cleavage produces “blunt” ends.Recognition sequences in DNA differ for each restriction enzyme,producing differences in the length, sequence and strand orientation (5′end or the 3′ end) of a sticky-end “overhang” of an enzyme restriction.Different restriction enzymes that recognize the same sequence are knownas neoschizomers. These often cleave in different locales of thesequence. Different enzymes that recognize and cleave in the samelocation are known as isoschizomers.

Protein: A protein typically comprises one or more peptides orpolypeptides. A protein is typically folded into 3-dimensional form,which may be required for the protein to exert its biological function.The sequence of a protein or peptide is typically understood to be theorder, i.e. the succession of its amino acids.

Recombinant protein: The term ‘recombinant protein’ refers to proteinsthat have been produced in a heterologous system, that is, in anorganism that naturally does not produce such a protein, or a variant ofsuch a protein. Alternatively, the organism may naturally produce theprotein, but in lower amounts so that the recombinant expressionincreases the amount of said protein. Typically, the heterologoussystems used in the art to produce recombinant proteins are bacteria(e.g., Escherichia coli), yeast (e.g., Saccharomyces cerevisiae) orcertain mammalian cell culture lines.

Plasmid DNA (vectors): The term ‘plasmid DNA’ or ‘plasmid DNA vector’refers to a circular nucleic acid molecule, preferably to an artificialnucleic acid molecule. A plasmid DNA in the context of the presentinvention is suitable for incorporating or harboring a desired nucleicacid sequence, such as a nucleic acid sequence comprising a sequenceencoding an RNA and/or an open reading frame encoding at least onepeptide or polypeptide. Such plasmid DNA constructs/vectors may bestorage vectors, expression vectors, cloning vectors, transfer vectorsetc. A storage vector is a vector, which allows the convenient storageof a nucleic acid molecule, for example, of an RNA molecule. Thus, theplasmid DNA may comprise a sequence corresponding (coding for), e.g., toa desired RNA sequence or a part thereof, such as a sequencecorresponding to the open reading frame and the 5′- and/or 3′UTR of anmRNA. An expression vector may be used for production of expressionproducts such as RNA, e.g. mRNA in a process called RNA in vitrotranscription. For example, an expression vector may comprise sequencesneeded for RNA in vitro transcription of a sequence stretch of thevector, such as a promoter sequence, e.g. an RNA promoter sequence,preferably T3, T7 or SP6 RNA promotor sequences. A cloning vector istypically a vector that contains a cloning site, which may be used toincorporate nucleic acid sequences (insert) into the vector. A cloningvector may be, e.g., a plasmid vector or a bacteriophage vector. Atransfer vector may be a vector, which is suitable for transferringnucleic acid molecules into cells or organisms, for example, viralvectors. Preferably, a plasmid DNA vector in the sense of the presentinvention comprises a multiple cloning site, an RNA promoter sequence,optionally a selection marker, such as an antibiotic resistance factor,and a sequence suitable for multiplication of the vector, such as anorigin of replication. Particularly preferred in the context of thepresent invention are plasmid DNA vectors, or expression vectors,comprising promoters for DNA-dependent RNA polymerases such as T3, T7and Sp6. As plasmid backbone, particularly preferred are pUC19 andpBR322.

Template DNA: As used herein, the term ‘template DNA’ (or ‘DNAtemplate’) typically relates to a DNA molecule comprising a nucleic acidsequence encoding the RNA sequence to be in vitro transcribed. Thetemplate DNA is used as template for in vitro transcription in order toproduce the RNA encoded by the template DNA. Therefore, the template DNAcomprises all elements necessary for in vitro transcription,particularly a promoter element for binding of a DNA dependent RNApolymerase as e.g. T3, T7 and SP6 RNA polymerases 5′ of the DNA sequenceencoding the target RNA sequence. Furthermore the template DNA maycomprise primer binding sites 5′ and/or 3′ of the DNA sequence encodingthe target RNA sequence to determine the identity of the DNA sequenceencoding the target RNA sequence e.g. by PCR or DNA sequencing. As usedherein, the term ‘template DNA’ may also refer to a DNA vector, such asa plasmid DNA, which comprises a nucleic acid sequence encoding the RNAsequence. Further, the ‘template DNA’ in the context of the presentinvention may be a linear or a circular DNA molecule.

Target Sequence: A ‘target sequence’ as used herein is typicallyunderstood as the sequence of the RNA, which is encoded by the nucleicacid sequence comprised in the template DNA. The target sequence is thusthe sequence to be synthesized by in vitro transcription, e.g. aprotein-coding sequence or another RNA as defined herein like is RNA,antisense RNA etc.

Linear template DNA plasmid: The linear template DNA plasmid is obtainedby contacting the plasmid DNA with a restriction enzyme under suitableconditions so that the restriction enzyme cuts the plasmid DNA at itsrecognition site(s) and disrupts the plasmid structure. This reaction iscalled linearization reaction. Hence, the linear template DNA comprisesa free 5′ end and a free 3′ end, which are not linked to each other. Ifthe plasmid DNA contains only one recognition site for the restrictionenzyme, the linear template DNA has the same number of nucleotides asthe plasmid DNA. If the plasmid DNA contains more than one recognitionsite for the restriction enzyme, the linear template DNA has a smallernumber of nucleotides than the plasmid DNA. The linear template DNA isthen the fragment of the plasmid DNA, which contains the elementsnecessary for RNA in vitro transcription, that is a promoter element forRNA transcription and the template DNA element. The DNA sequenceencoding the target RNA sequence of the linear template DNA determinesthe sequence of the transcribed RNA by the rules of base-pairing.5′-cap: A 5′-cap is an entity, typically a modified nucleotide entity,which generally “caps” the 5′-end of a mature mRNA. A 5′-cap maytypically be formed by a modified nucleotide (cap analog), particularlyby a derivative of a guanine nucleotide. Preferably, the 5′-cap islinked to the 5′-terminus via a 5′-5′-triphosphate linkage. A 5′-cap maybe methylated, e.g. m7GpppN (e.g. m7G(5′)ppp(5′)G (m7G)), wherein N isthe terminal 5′ nucleotide of the nucleic acid carrying the 5′-cap,typically the 5′-end of an RNA. Further examples of 5′cap structuresinclude glyceryl, inverted deoxy abasic residue (moiety), 4′,5′methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,L-nucleotides, alpha-nucleotide, modified base nucleotide,threo-pentofuranosyl nucleotide, acyclic 3′,4′-seco nucleotide, acyclic3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety, 3′-3′-inverted abasic moiety,3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety,1,4-butanediol phosphate, 3′-phosphoramidate, hexylphosphate, aminohexylphosphate, 3′-phosphate, 3′phosphorothioate, phosphorodithioate, orbridging or non-bridging methylphosphonate moiety. Further modified5′-CAP structures which may be used in the context of the presentinvention are CAP1 (methylation of the ribose of the adjacent nucleotideof m7GpppN), CAP2 (methylation of the ribose of the 2nd nucleotidedownstream of the m7GpppN), CAP3 (methylation of the ribose of the 3rdnucleotide downstream of the m7GpppN), CAP4 (methylation of the riboseof the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse CAPanalogue, modified ARCA (e.g. phosphothioate modified ARCA), inosine,N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine.

Poly(A) sequence: A poly(A) sequence, also called poly(A) tail or3′-poly(A) tail, is typically understood to be a sequence of adeninenucleotides, e.g., of up to about 400 adenine nucleotides, e.g. fromabout 20 to about 400, preferably from about 50 to about 400, morepreferably from about 50 to about 300, even more preferably from about50 to about 250, most preferably from about 60 to about 250 adeninenucleotides. A poly(A) sequence is typically located at the 3′end of anmRNA. In the context of the present invention, a poly(A) sequence may belocated within an mRNA or any other nucleic acid molecule, such as,e.g., in a vector, for example, in a vector serving as template for thegeneration of an RNA, preferably an mRNA, e.g., by transcription of thevector.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotide monomers.These nucleotides are usually adenosine-monophosphate,uridine-monophosphate, guanosine-monophosphate andcytidine-monophosphate monomers, which are connected to each other alonga so-called backbone. The backbone is formed by phosphodiester bondsbetween the sugar, i.e. ribose, of a first and a phosphate moiety of asecond, adjacent monomer. The specific order of the monomers, i.e. theorder of the bases linked to the sugar/phosphate-backbone, is called theRNA-sequence. Usually RNA may be obtainable by transcription of aDNA-sequence, e.g., inside a cell. In eukaryotic cells, transcription istypically performed inside the nucleus or the mitochondria. In vivo,transcription of DNA usually results in the so-called premature RNA,which has to be processed into so-called messenger-RNA, usuallyabbreviated as mRNA. Processing of the premature RNA, e.g. in eukaryoticorganisms, comprises a variety of differentposttranscriptional-modifications such as splicing, 5′-capping,polyadenylation, export from the nucleus or the mitochondria and thelike. The sum of these processes is also called maturation of RNA. Themature messenger RNA usually provides the nucleotide sequence that maybe translated into an amino acid sequence of a particular peptide orprotein. Typically, a mature mRNA comprises a 5′-cap, optionally a5′UTR, an open reading frame, optionally a 3′UTR and a poly(A) sequence.Aside from messenger RNA, several non-coding types of RNA exist whichmay be involved in regulation of transcription and/or translation, andimmunostimulation. The term “RNA” further encompass other coding RNAmolecules, such as viral RNA, retroviral RNA and replicon RNA, smallinterfering RNA (siRNA), antisense RNA, CRISPR RNA, ribozymes, aptamers,riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA(rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA),microRNA (miRNA), and Piwi-interacting RNA (piRNA).

5′-untranslated region (5′-UTR): As used herein, the term ‘5′-UTR’typically refers to a particular section of messenger RNA (mRNA). It islocated 5′ of the open reading frame of the mRNA. Typically, the 5′-UTRstarts with the transcriptional start site and ends one nucleotidebefore the start codon of the open reading frame. The 5′-UTR maycomprise elements for controlling gene expression, also calledregulatory elements. Such regulatory elements may be, for example,ribosomal binding sites or a 5′-Terminal Oligopyrimidine Tract. The5′-UTR may be posttranscriptionally modified, for example by addition ofa 5′-CAP. In the context of the present invention, a 5′-UTR correspondsto the sequence of a mature mRNA, which is located between the 5′-CAPand the start codon. Preferably, the 5′-UTR corresponds to the sequence,which extends from a nucleotide located 3′ to the 5′-CAP, preferablyfrom the nucleotide located immediately 3′ to the 5′-CAP, to anucleotide located 5′ to the start codon of the protein coding region,preferably to the nucleotide located immediately 5′ to the start codonof the protein coding region. The nucleotide located immediately 3′ tothe 5′-CAP of a mature mRNA typically corresponds to the transcriptionalstart site. The term “corresponds to” means that the 5′-UTR sequence maybe an RNA sequence, such as in the mRNA sequence used for defining the5′-UTR sequence, or a DNA sequence, which corresponds to such RNAsequence. In the context of the present invention, the term “a 5′-UTR ofa gene”, such as “a 5′-UTR of a TOP gene”, is the sequence, whichcorresponds to the 5′-UTR of the mature mRNA derived from this gene,i.e. the mRNA obtained by transcription of the gene and maturation ofthe pre-mature mRNA. The term “5′-UTR of a gene” encompasses the DNAsequence and the RNA sequence of the 5′-UTR. Preferably, the 5′-UTR usedaccording to the present invention is heterologous to the coding regionof the mRNA sequence. Even if 5′-UTR's derived from naturally occurringgenes are preferred, also synthetically engineered UTR's may be used inthe context of the present invention.

3′-untranslated region (3′-UTR): In the context of the presentinvention, a 3′-UTR is typically the part of an mRNA, which is locatedbetween the protein coding region (i.e. the open reading frame) and the3′-terminus of the mRNA. A 3′-UTR of an mRNA is not translated into anamino acid sequence. The 3′-UTR sequence is generally encoded by thegene, which is transcribed into the respective mRNA during the geneexpression process. In the context of the present invention, a 3′-UTRcorresponds to the sequence of a mature mRNA, which is located 3′ to thestop codon of the protein coding region, preferably immediately 3′ tothe stop codon of the protein coding region, and which extends to the5′-side of the 3′-terminus of the mRNA or of the poly(A) sequence,preferably to the nucleotide immediately 5′ to the poly(A) sequence. Theterm “corresponds to” means that the 3′-UTR sequence may be an RNAsequence, such as in the mRNA sequence used for defining the 3′-UTRsequence, or a DNA sequence, which corresponds to such RNA sequence. Inthe context of the present invention, the term “a 3′-UTR of a gene”,such as “a 3′-UTR of an albumin gene”, is the sequence, whichcorresponds to the 3′-UTR of the mature mRNA derived from this gene,i.e. the mRNA obtained by transcription of the gene and maturation ofthe pre-mature mRNA. The term “3′-UTR of a gene” encompasses the DNAsequence and the RNA sequence of the 3′-UTR. Preferably, the 3′-UTR usedaccording to the present invention is heterologous to the coding regionof the mRNA sequence. Even if 3′-UTR's derived from naturally occurringgenes are preferred, also synthetically engineered UTR's may be used inthe context of the present invention.

In vitro transcribed RNA: An in vitro transcribed RNA is an RNA moleculethat has been synthesized from a template DNA, commonly a linearized andpurified plasmid template DNA, a PCR product, or an oligonucleotide. RNAsynthesis occurs in a cell free (“in vitro”) assay catalyzed by DNAdependent RNA polymerases. In a process called RNA in vitrotranscription, virtually all nucleotides analogues into RNA. Particularexamples of DNA dependent RNA polymerases are the T7, T3, and SP6 RNApolymerases. An in vitro transcribed RNA may comprise elements such as5′-cap, optionally a 5′UTR, an open reading frame, optionally a 3′UTRand a poly(A) sequence. Aside from proteinogenic messenger RNA, severalnon-coding types of RNA exist which may be involved in regulation oftranscription and/or translation. Such All RNA molecules as definedherein may also be synthesized by RNA in vitro transcription.

RNA species: The term “RNA species” denotes a group of the same RNAmolecules which are similar in their RNA sequence and/or their sequencelength. Hence, the RNA molecules within one RNA species are encoded bythe same template DNA. If the RNA present within the sample is a codingRNA, one RNA species encodes one target peptide or protein.

DNA: DNA is the usual abbreviation for deoxy-ribonucleic-acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotide monomers.These nucleotides are usually deoxy-adenosine-monophosphate,deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate anddeoxy-cytidine-monophosphate monomers which are—by themselves—composedof a sugar moiety (deoxyribose), a base moiety and a phosphate moiety,and polymerise by a characteristic backbone structure. The backbonestructure is, typically, formed by phosphodiester bonds between thesugar moiety of the nucleotide, i.e. deoxyribose, of a first and aphosphate moiety of a second, adjacent monomer. The specific order ofthe monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the DNA-sequence. DNA may besingle-stranded or double-stranded. In the double stranded form, thenucleotides of the first strand typically hybridize with the nucleotidesof the second strand, e.g. by A/T-base-pairing and G/C-base-pairing.

Cloning site, multiple cloning site: A cloning site is typicallyunderstood to be a segment of a nucleic acid molecule, which is suitablefor insertion of a nucleic acid sequence, e.g., a nucleic acid sequencecomprising an open reading frame. Insertion may be performed by anymolecular biological method known to the one skilled in the art, e.g. byrestriction and ligation. A cloning site typically comprises one or morerestriction enzyme recognition sites (restriction sites). These one ormore restrictions sites may be recognized by restriction enzymes whichcleave the DNA at these sites. A cloning site which comprises more thanone restriction site may also be termed a multiple cloning site (MCS) ora polylinker.

Open reading frame: An open reading frame (ORF) in the context of theinvention may typically be a sequence of several nucleotide triplets,which may be translated into a peptide or protein. An open reading framepreferably contains a start codon, i.e. a combination of threesubsequent nucleotides coding usually for the amino acid methionine(ATG), at its 5′-end and a subsequent region, which usually exhibits alength which is a multiple of 3 nucleotides. An ORF is preferablyterminated by a stop-codon (e.g., TAA, TAG, TGA). Typically, this is theonly stop-codon of the open reading frame. Thus, an open reading framein the context of the present invention is preferably a nucleotidesequence, consisting of a number of nucleotides that may be divided bythree, which starts with a start codon (e.g. ATG) and which preferablyterminates with a stop codon (e.g., TAA, TGA, or TAG). The open readingframe may be isolated or it may be incorporated in a longer nucleic acidsequence, for example in a vector or an mRNA. An open reading frame mayalso be termed “protein coding region”.

RNA in vitro transcription: The term “RNA in vitro transcription” (or‘in vitro transcription’) relates to a process wherein RNA, inparticular mRNA, is synthesized in a cell-free system (in vitro).Preferably, cloning vectors DNA, particularly plasmid DNA vectors areapplied as template for the generation of RNA transcripts. These cloningvectors are generally designated as transcription vector. RNA may beobtained by DNA dependent in vitro transcription of an appropriate DNAtemplate, which according to the present invention is preferably alinearized plasmid DNA template. The promoter for controlling RNA invitro transcription can be any promoter for any DNA dependent RNApolymerase. Particular examples of DNA dependent RNA polymerases are theT7, T3, and SP6 RNA polymerases. A DNA template for RNA in vitro RNAtranscription may be obtained by cloning of a nucleic acid, inparticular cDNA corresponding to the respective RNA to be in vitrotranscribed, and introducing it into an appropriate vector for RNA invitro transcription, for example in plasmid circular plasmid DNA. ThecDNA may be obtained by reverse transcription of mRNA or chemicalsynthesis. Moreover, the DNA template for in vitro RNA synthesis mayalso be obtained by gene synthesis. Preferably cloning vectors are usedfor RNA in vitro RNA transcription, which are generally designatedtranscription vectors.

Kozak sequence: As used herein, the term ‘Kozak sequence’ typicallyrefers to a sequence on an mRNA molecule, which is recognized by theribosome as the translational start site of a protein encoded by thatmRNA molecule. In a preferred embodiment, that sequence may comply witha consensus sequence for a nucleotide sequence mediating initiation oftranslation, preferably with the consensus sequence (gcc)gccRccAUGG,wherein a lower case letter denotes the most common base at a positionwhere the base can nevertheless vary; upper case letters indicate highlyconserved bases, ‘AUGG’; ‘R’ indicates that a purine (adenine orguanine, preferably adenine) is present at this position; and thesequence in brackets is of uncertain significance.

HPLC: High-performance liquid chromatography (HPLC; formerly referred toas high-pressure liquid chromatography), is a technique in analyticchemistry used to separate the components in a mixture, to identify eachcomponent, and to quantify each component. It relies on pumps to pass apressurized liquid solvent containing the sample mixture through acolumn filled with a solid adsorbent material. Each component in thesample interacts slightly differently with the adsorbent material,causing different flow rates for the different components and leading tothe separation of the components as they flow out the column. HPLC isdistinguished from traditional (“low pressure”) liquid chromatographybecause operational pressures are significantly higher (50-350 bar),while ordinary liquid chromatography typically relies on the force ofgravity to pass the mobile phase through the column. Due to the smallsample amount separated in analytical HPLC, typical column dimensionsare 2.1-4.6 mm diameter, and 30-250 mm length. Also HPLC columns aremade with smaller sorbent particles (2-50 micrometer in average particlesize). This gives HPLC superior resolving power when separatingmixtures, which is why it is a popular chromatographic technique. Theschematic of an HPLC instrument typically includes a sampler, pumps, anda detector. The sampler brings the sample mixture into the mobile phasestream which carries it into the column. The pumps deliver the desiredflow and composition of the mobile phase through the column. Thedetector generates a signal proportional to the amount of samplecomponent emerging from the column, hence allowing for quantitativeanalysis of the sample components. A digital microprocessor and usersoftware control the HPLC instrument and provide data analysis. Somemodels of mechanical pumps in a HPLC instrument can mix multiplesolvents together in ratios changing in time, generating a compositiongradient in the mobile phase. Various detectors are in common use, suchas UV/Vis, photodiode array (PDA) or based on mass spectrometry. MostHPLC instruments also have a column oven that allows for adjusting thetemperature the separation is performed at.

Lyophilization: Freeze-drying, also known as lyophilization, orcryodesiccation, is a dehydration process typically used to preserve aperishable material or make the material more convenient for transport.Freeze-drying works by freezing the material and then reducing thesurrounding pressure to allow the frozen water in the material tosublimate directly from the solid phase to the gas phase.

Conditioning: the term “conditioning” comprises providing a compound,such as RNA or DNA, in a suitable form, e.g. a suitable buffer orsolvent, suitable concentration or suitable biochemical or biophysicalproperties. The conditioning may for example be performed using TFF,e.g. in a concentration and/or diafiltration step. Therefore,conditioning includes concentration and exchange of buffer or solvent bydiafiltration.

Purification: as used herein, the term “purification” or “purifying” or“pure” is understood to mean that the desired RNA or DNA in a sample isseparated and/or isolated from impurities, intermediates, byproductsand/or reaction components present therein or that the impurities,intermediates, byproducts and/or reaction components are at leastdepleted from the sample comprising the RNA or DNA. Non-limitingexamples of undesired constituents of RNA- or DNA-containing sampleswhich therefore need to be depleted may comprise degraded fragments orfragments which have arisen as a result of premature termination oftranscription, or also excessively long transcripts if plasmids are notcompletely linearized. Furthermore, intermediates may be depleted fromthe sample such as e.g. template DNA. Additionally, reaction componentssuch as enzymes, proteins, bacterial DNA and RNA, small molecules suchas spermidine, buffer components etc. may have to be depleted from theRNA/DNA sample. In addition, impurities such as, organic solvents, andnucleotides or other small molecules may be separated. Ideally, the RNAhas a higher purity and/or integrity after purification than thestarting material. The purity may be determined by methods commonlyknown to the skilled person, e.g. by gas chromatography, quantitativePCR, analytical HPLC or gel electrophoresis.

Tangential Flow Filtration (TFF) or Crossflow Filtration: Crossflowfiltration (also known as tangential flow filtration) is a type offiltration. Crossflow filtration is different from dead-end filtrationin which the feed is passed through a membrane or bed, the solids beingtrapped in the filter and the filtrate being released at the other end.Cross-flow filtration gets its name because the majority of the feedflow travels tangentially across the surface of the filter, rather thaninto the filter. The principal advantage of this is that the filter cake(which can blind the filter) is substantially washed away during thefiltration process, increasing the length of time that a filter unit canbe operational. It can be a continuous process, unlike batch-wisedead-end filtration. This type of filtration is typically selected forfeeds containing a high proportion of small particle size solids (wherethe permeate is of most value) because solid material can quickly block(blind) the filter surface with dead-end filtration. Applied pressurecauses one portion of the flow stream to pass through the membrane(filtrate/permeate) while the remainder (retentate) is recirculated backto the feed reservoir. The general working principle of TFF can be foundin literature, see e.g. Fernandez et al. (A BIOTECHNOLOGICA, Bd. 12,1992, Berlin, Pages 49-56) or Rathore, A S et al (Prep BiochemBiotechnol. 2011; 41(4):398-421). Further, the principle of TFF isillustrated in FIG. 1 . The primary applications for TFF areconcentration, diafiltration (desalting and buffer/solvent exchange),and fractionation of large from small biomolecules. Membranes withdifferent molecular weight cutoffs (MWCO) may be used for TFF. In thecontext of the present invention ultrafiltration membranes arepreferably used for TFF.

Two basic filter configurations are generally used for TFF. In cartridgefilters (often called hollow fiber filters), the membrane forms a set ofparallel hollow fibers. The feed stream passes through the lumen of thefibers and the permeate is collected from outside the fibers. Cartridgesare characterized in terms of fiber length, lumen diameter and number offibers, as well as filter pore size. In cassette filters, several flatsheets of membrane are held apart from each other and from the cassettehousing by support screens. The feed stream passes into the spacebetween two sheets and permeate is collected from the opposite side ofthe sheets. Cassettes are characterized in terms of flow path length andchannel height, as well as membrane pore size. The channel height isdetermined by the thickness of the support screen. Both cartridges andcassettes are constructed from materials chosen for mechanical strength,chemical and physical compatibility, and low levels of extractableand/or toxic compounds.

Ultrafiltration: Ultrafiltration is a filtration method using a membranein which forces like pressure or concentration gradients lead to aseparation through a semipermeable membrane. Suspended solids andsolutes of high molecular weight are retained in the so-calledretentate, while water and low molecular weight solutes pass through themembrane in the permeate. This separation process is used in industryand research for purifying and concentrating macromolecular (10³-10⁶ Da)solutions. Ultrafiltration is not fundamentally different frommicrofiltration. Both of these separate based on size exclusion orparticle capture. Ultrafiltration membranes are defined by the molecularweight cut-off (MWCO) of the membrane of between 2 and 100 nm (whichcorresponds to a MWCO between 1 and 1000 kDa). Ultrafiltration isapplied in cross-flow or dead-end mode.

Feed: Material or solution that is fed into the filter, for example thelinearization reaction or the RNA in vitro transcription reactionmixture.

Transfer RNA: A transfer RNA is an adaptor molecule composed of RNA,typically 76 to 90 nucleotides in length that serves as the physicallink between the nucleotide sequence of nucleic acids (DNA and RNA) andthe amino acid sequence of proteins. It does this by carrying an aminoacid to the protein synthetic machinery of a cell (ribosome) as directedby a three-nucleotide sequence (codon) in a messenger RNA (mRNA). Assuch, tRNAs are a necessary component of protein translation, thebiological synthesis of new proteins according to the genetic code. Thestructure of tRNA can be decomposed into its primary structure, itssecondary structure (usually visualized as the cloverleaf structure),and its tertiary structure (all tRNAs have a similar L-shaped 3Dstructure that allows them to fit into the P and A sites of theribosome). The cloverleaf structure becomes the 3D L-shaped structurethrough coaxial stacking of the helices, which is a common RNA tertiarystructure motif.

Ribosomal RNA (rRNA): the RNA component of the ribosome; rRNA isessential for protein synthesis in all living organisms. It constitutesthe predominant material within the ribosome, which is approximately 60%rRNA and 40% protein by weight. Ribosomes contain two major rRNAs and 50or more proteins. The ribosomal RNAs form two subunits, the largesubunit (LSU) and small subunit (SSU). The LSU rRNA acts as a ribozyme,catalyzing peptide bond formation, mRNA is sandwiched between the smalland large subunits, and the ribosome catalyzes the formation of apeptide bond between the two amino acids that are contained in the rRNA.

Viral RNA: viral RNA is RNA derived from a virus, for example aretrovirus. It may be directly translated into the desired viralproteins. Portions of the viral RNA may be skipped during translation.The result is that many different proteins can be created from the samemRNA strand, with similar 5′ ends (to varying degrees) and same 3′ ends.Or, different proteins can be created from positive sense viral RNA andnegative sense viral RNA.

Replicons: A replicon is an RNA molecule, or a region of an RNA, thatreplicates from a single origin of replication.

Antisense-RNA: Antisense RNA (asRNA) or mRNA-interfering complementaryRNA (micRNA) is a single-stranded RNA that is complementary to a portionof an mRNA strand transcribed within a cell. Antisense RNA may forexample be introduced into a cell to inhibit translation of acomplementary mRNA by base pairing to it and physically obstructing thetranslation machinery.

Immunostimulating RNA (isRNA): An immunostimulating RNA (isRNA) in thecontext of the invention may typically be a RNA that is able to inducean innate immune response itself. It usually does not have an openreading frame and thus does not provide a peptide-antigen or immunogenbut elicits an innate immune response e.g. by binding to a specific kindof Toll-like-receptor (TLR) or other suitable receptors. However, ofcourse also mRNAs having an open reading frame and coding for apeptide/protein (e.g. an antigenic function) may induce an innate immuneresponse.

Small interfering RNA (siRNA): also known as short interfering RNA orsilencing RNA, is a class of double-stranded RNA molecules, 20-25 basepairs in length. siRNA plays many roles, but it is most notable in theRNA interference (RNAi) pathway, where it interferes with the expressionof specific genes with complementary nucleotide sequences. siRNAfunctions by causing mRNA to be broken down after transcription,resulting in no translation. siRNA also acts in RNAi-related pathways,e.g., as an antiviral mechanism or in shaping the chromatin structure ofa genome. siRNAs have a well-defined structure: a short (usually 20 to24-bp) double-stranded RNA (dsRNA) with phosphorylated 5′ ends andhydroxylated 3′ ends with two overhanging nucleotides. The Dicer enzymecatalyzes production of siRNAs from long dsRNAs and small hairpin RNAs.

Ribozyme: Ribozymes are ribonucleic acid enzymes, also termed catalyticRNA. Ribozymes are RNA molecules that are capable of catalyzing specificbiochemical reactions, similar to the action of protein enzymes.Ribozymes have diverse structures and mechanisms. Examples of ribozymesinclude the hammerhead ribozyme, the VS ribozyme, Leadzyme and thehairpin ribozyme.

Aptamers: Aptamers (from the Latin aptus—fit, and Greek meros—part) areRNA-based oligonucleotide molecules that bind to a specific targetmolecule. Aptamers are usually created by selecting them from a largerandom sequence pool, but natural aptamers also exist in riboswitches.Aptamers also include RNA-based oligonucleotide molecules that bind to aspecific target molecule that are combined with ribozymes to self-cleavein the presence of their target molecule.

CRISPR (clustered regularly interspaced short palindromic repeats):segments of prokaryotic DNA containing short repetitions of basesequences. Each repetition is followed by short segments of “spacer DNA”from previous exposures to a bacterial virus or plasmid. CRISPRs arefound in approximately 40% of sequenced bacteria genomes and 90% ofsequenced archaea. CRISPRs are often associated with cas genes that codeproteins related to CRISPRs. The CRISPR/Cas system is a prokaryoticimmune system that confers resistance to foreign genetic elements suchas plasmids and phages and provides a form of acquired immunity. CRISPRspacers recognize and cut these exogenous genetic elements in a manneranalogous to RNAi in eukaryotic organisms.

Piwi-interacting RNA: the largest class of small non-coding RNAmolecules expressed in animal cells. piRNAs form RNA-protein complexesthrough interactions with piwi proteins. These piRNA complexes have beenlinked to both epigenetic and post-transcriptional gene silencing ofretrotransposons and other genetic elements in germ line cells,particularly those in spermatogenesis. They are distinct from microRNA(miRNA) in size (26-31 nt rather than 21-24 nt), lack of sequenceconservation, and increased complexity. piRNAs have been identified inboth vertebrates and invertebrates, and although biogenesis and modes ofaction do vary somewhat between species, a number of features areconserved. piRNAs have no clear secondary structure motifs, the lengthof a piRNA is between 26 and 31 nucleotides, and the bias for a 5′uridine is common to piRNAs in both vertebrates and invertebrates.piRNAs are found in clusters throughout the genome; these clusters maycontain as few as ten or up to many thousands of piRNAs and can vary insize from one to one hundred kb.

RNA modification: The term “RNA modification” as used herein may referto chemical modifications comprising backbone modifications as well assugar modifications or base modifications.

In this context, a modified RNA molecule as defined herein may containnucleotide analogues/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in an RNAmolecule as defined herein are chemically modified. A sugar modificationin connection with the present invention is a chemical modification ofthe sugar of the nucleotides of the RNA molecule as defined herein.Furthermore, a base modification in connection with the presentinvention is a chemical modification of the base moiety of thenucleotides of the RNA molecule. In this context, nucleotide analoguesor modifications are preferably selected from nucleotide analogues,which are applicable for transcription and/or translation.

Sugar Modifications: The modified nucleosides and nucleotides, which maybe incorporated into a modified RNA molecule as described herein, can bemodified in the sugar moiety. For example, the 2′ hydroxyl group (OH)can be modified or replaced with a number of different “oxy” or “deoxy”substituents. Examples of “oxy”-2′ hydroxyl group modifications include,but are not limited to, alkoxy or aryloxy (—OR, e.g., R═H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols(PEG), —O(CH₂CH₂O)nCH₂CH₂OR; “locked” nucleic acids (LNA) in which the2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbonof the same ribose sugar; and amino groups (—O-amino, wherein the aminogroup, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl,arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylenediamine, polyamino) or aminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA molecule can include nucleotidescontaining, for instance, arabinose as the sugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides, which may be incorporated into a modifiedRNA molecule as described herein. The phosphate groups of the backbonecan be modified by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleosides and nucleotidescan include the full replacement of an unmodified phosphate moiety witha modified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified RNA molecule as described herein can further be modified inthe nucleobase moiety. Examples of nucleobases found in RNA include, butare not limited to, adenine, guanine, cytosine and uracil. For example,the nucleosides and nucleotides described herein can be chemicallymodified on the major groove face. In some embodiments, the major groovechemical modifications can include an amino group, a thiol group, analkyl group, or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-fluorothymidine-5′-triphosphate,2′-O-methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-bromo-2′-deoxycytidine-5′-triphosphate,5-bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-propynyl-2′-deoxycytidine-5′-triphosphate,5-propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine,5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine or5′-0-(1-thiophosphate)-pseudouridine.

In further specific embodiments, a modified RNA may comprise nucleosidemodifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine,6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine,8-azido-adenosine, 7-deaza-adenosine.

Sequence Modifications:

The term “RNA modification” as used herein may further refer to an RNAwhich is modified in regards to the nucleotide sequence compared to thewild type sequence. Particularly if the RNA is a coding RNA, e.g. anmRNA the RNA may be sequence modified in the coding region (open readingframe). On the one hand, the G/C content of the region of the modifiedRNA coding for a peptide or polypeptide may be greater than the G/Ccontent of the coding region of the wild-type RNA coding for the peptideor polypeptide, the coded amino acid sequence being unchanged relativeto the wild-type. This modification is based on the fact that thesequence order of the RNA domain to be translated is essential forefficient RNA translation. In this respect, the composition and sequenceof the various nucleotides has an important part to play. In particular,sequences with an elevated G(guanosine)/C(cytosine) content are morestable than sequences with an elevated A(adenosine)/U(uracil) content.Thus, according to the invention, while retaining the translated aminoacid sequence, the codons are varied relative to the wild-type RNA insuch a manner that they have a greater content of G/C nucleotides. Sinceseveral codons code for one and the same amino acid (degeneration of thegenetic code), it is possible to determine the codons which are mostfavourable for stability (alternative codon usage). On the other hand itis also possible to provide a translation-optimised RNA by sequencemodification.

Concentration: Concentration is a simple process that involves removingfluid from a solution while retaining the solute molecules. Theconcentration of the solute increases in direct proportion to thedecrease in solution volume, i.e. halving the volume effectively doublesthe concentration of the solute molecules.

Diafiltration: Diafiltration is the fractionation process that washessmaller molecules through a membrane and leaves larger molecules in theretentate without ultimately changing concentration. It can be used toremove salts or exchange buffers or solvents to deliver the product inthe desired buffer or solvent. In the context of the present invention,diafiltration against water (WFI) and/or NaCl solution is performed inorder to remove LMWC and HMWC (e.g. salts, short oligonucleotides, smallproteins, spermidine and organic solvents). Diafiltration can beperformed either discontinuously or alternatively, continuously. Incontinuous diafiltration, the diafiltration solution is added to thesample feed reservoir at the same rate as filtrate is generated. In thisway, the volume in the sample reservoir remains constant but the smallmolecules (e.g. salts) that can freely permeate through the membrane arewashed away. Using salt removal as an example, each additionaldiafiltration volume (DV) reduces the salt concentration further. Indiscontinuous diafiltration, the solution is first diluted and thenconcentrated back to the starting volume. This process is then repeateduntil the required concentration of small molecules (e.g. salts)remaining in the reservoir is reached. Each additional diafiltrationvolume (DV) reduces the salt concentration further. Continuousdiafiltration requires less diafiltration volume to achieve the samedegree of salt reduction as discontinuous diafiltration.

Diafiltration volume (DV): a single diafiltration volume is the volumeof retentate when diafiltration is started.

deltaP (dp): Describes the pressure difference of the feed pressure (μl)and the retentate pressure (p2) in TFF. The value of dp is directlyproportional to the flow rate over the membrane.

Feed pressure: The pressure measured at the inlet port of acartridge/hollow fibre or cassette.

Feed flow rate: Volume of feed solution loaded on the filter membraneper given membrane area during a given time.

Transmembrane pressure (TMP): the driving force for liquid transportthrough the ultrafiltration membrane. Calculated as the average pressureapplied to the membrane minus any filtrate pressure. In most cases,pressure at filtrate port equals zero.

Filtrate or Permeate: the portion of sample that has flowed through themembrane.

Flux: Flux represents the volume of solution flowing through a givenmembrane area during a given time. Expressed as LMH (liters per squaremeter per hour).

Permeate flux rate: permeate flow divided by the effective membrane area(L/h/m²).

Permeate flow: flow rate of the permeate (L/h).

Membrane load: Amount of product (linearized plasmid DNA or in vitrotranscribed RNA) per surface area of the filter membrane.

RNA integrity: The relative RNA integrity is preferably determined asthe percentage of full-length RNA (i.e. non-degraded RNA) with respectto the total amount of RNA (i.e. full-length RNA and degraded RNAfragments (which appear as smears in gel electrophoresis)). RNAintegrity can be measured and quantified by agrose gel electrophoresis(AGE) and/or analytical RP-HPLC.

Molecular Weight Cut Off (MWCO): The molecular weight cutoff of amembrane, sometimes called Nominal Molecular Weight Limit (NMWL), isdefined by its ability to retain a given percentage of a globular soluteof a defined molecular weight. Solute retention can however vary due tomolecular shape, structure, solute concentration, presence of othersolutes and ionic conditions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for producing and purifyingRNA, comprising the steps of

A) providing DNA encoding the RNA;

B) transcription of the DNA to yield a solution comprising transcribedRNA; and

C) conditioning and/or purifying of the solution comprising transcribedRNA by one or more steps of TFF.

The individual steps of the methods according to the present inventionmay be performed sequentially or they may at least partially overlap.

The RNA which is to be purified with the method according to theinvention is a ribonucleic acid of any type, preferably as definedherein. The RNA is particularly preferably selected from mRNA, viralRNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA),antisense RNA, clustered regularly interspaced short palindromic repeats(CRISPR) RNA, ribozymes, aptamers, riboswitches, immunostimulating RNA,transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA),small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA(piRNA) or whole-cell RNA (total RNA extract). The RNA to be isolatedmay be single-stranded or double-stranded. Single-stranded RNA mayoptionally form secondary structures by refolding, the RNA to beseparated typically being single-stranded. The RNA may be unlabelled oralso labelled (with a fluorescent label or a radiolabel or an antibodyepitope). One example of a labelled RNA is digoxigenin-labelled RNA. TheRNA may also contain modifications (modified nucleotides), preferably asdefined herein.

In a preferred embodiment of the method according to the invention, theRNA to be separated has a size of up to about 15000 nucleotides (assingle stranded RNA molecule) or base pairs (as double stranded RNAmolecule), in particular 100 to 10000, more preferably 500 to 10000nucleotides or base pairs, even more preferably 500 to 7000 nucleotidesor base pairs and even more preferably 500 to 5000 nucleotides or basepairs. For this size of RNA, it has proved possible to achieve very goodresults with regard to purification of the RNA, since the methodaccording to the invention is particularly well suited to RNA of thissize. Optionally, however, smaller RNA fragments, for example with alength of 30-500, 50-500 or 100-500 or 20-200, 20-100, 20-50 or 20-30nucleotides may also be separated in this way.

If the RNA to be separated is a coding RNA, e.g. an mRNA, viral RNA orreplicon RNA, it will preferably code for peptides or proteins. The RNAmay encode a protein sequence or a fragment or variant thereof (e.g.fusion proteins), preferably selected from therapeutically activeproteins or peptides, including adjuvant proteins, tumor antigens,cancer antigens, pathogenic antigens (e.g. selected, from animalantigens, from viral antigens, from protozoal antigens, from bacterialantigens), allergenic antigens, autoimmune antigens, or furtherantigens, allergens, antibodies, immunostimulatory proteins or peptides,antigen-specific T-cell receptors, biologicals, cell penetratingpeptides, secreted proteins, plasma membrane proteins, cytoplasmic orcytoskeletal proteins, intracellular membrane bound proteins, nuclearproteins, proteins associated with human disease, targeting moieties orthose proteins encoded by the human genome, for which no therapeuticindication has been identified but which nonetheless have utility inareas of research and discovery.

In a preferred embodiment, the RNA obtained according to the method ofthe present invention may be used as a pharmaceutical composition or maybe a component of a pharmaceutical composition together with additionalcomponents.

The steps of the method according to the present invention will bedescribed in the following in more detail.

Step A:

In a preferred embodiment, the DNA encoding the RNA provided in step A)may be any kind of DNA, including plasmid DNA, genomic DNA, or DNAfragments such as DNA-fragments obtained by polymerase chain reaction(PCR).

In a preferred embodiment, the DNA provided in step A) is plasmid DNA.The plasmid DNA may be provided in a circular form or in a linearizedform. The linearized form is preferred and may be obtained, e.g. bydigestion with a restriction enzyme (linearization). Preferredrestriction enzymes are BciVI (BfuI), BcuI (SpeI), EcoRI, AatII, AgeI(BshTI), ApaI, BamHI, BglII, BlpI (Bpu1102I), BsrGI (Bsp1407), ClaI(Bsu15I), EcoRI, EcoRV (Eco32I), HindIII, KpnI, MluI, NcoI, NdeI, NheI,NotI, NsiI, Mph1103I), PstI, PvuI, PvuII, SacI, SalI, ScaI, SpeI, XbaI,XhoI, SacII (Cfr42I), XbaI. The skilled artisan knows under whatconditions the linearization of the DNA may be performed and that thelinearization conditions are dependent on which kind of restrictionenzyme is used. In one embodiment, the plasmid DNA may be conditioned orpurified by using one or more steps of TFF prior to linearization.

The at least one step of TFF may comprise at least one diafiltrationstep and/or at least one concentration step. The diafiltration andconcentration step may be performed separately but they may also atleast partially overlap. The at least one or more steps of TFF mayefficiently remove contaminants, such as HMWC and LMWC, e.g. DNAfragments, organic solvents, buffer components such as salts anddetergents. The use of TFF may thus reduce or abolish the need to purifythe DNA by means of organic solvent extraction such as phenol/chloroformextraction and/or alcohol precipitation of nucleic acids such as RNA andDNA, e.g. by high salt/alcohol precipitation such as NaCl/isopropanolprecipitation. Further, it was found that, surprisingly, the use of TFFdoes not negatively affect the stability of the DNA, e.g. due to shearstress during pumping.

In preferred embodiments the one or more steps of TFF priorlinearization is performed as described for step A3) conditioning,and/or purifying of the linearized DNA by one or more steps of TFF.

In a preferred embodiment, the linearization reaction comprises:

1 μg plasmid DNA;

0.5 μl reaction buffer;

3 Units restriction enzyme;

Add. 5 μl with WFI (water for injection).

The reaction is preferably incubated for 4 to 5 hour at 37° C.

In a further preferred embodiment which is useful for large-scaleproduction, the linearization reaction comprises:

30 mg plasmid DNA;

15 ml reaction buffer (10× restriction buffer)

9 ml restriction enzyme [10 U/μl]

The reaction is filled up to a final volume of 150 ml using WFI andincubated for 4 to 5 hour at 37° C.

In a preferred embodiment, the linearization reaction, e.g. thelinearization reaction using a restriction enzyme, may be terminated.The termination of the linearization may be performed by adding an agentthat inhibits the activity of the restriction enzyme. In one embodiment,the termination of linearization may be performed by adding an effectiveamount of ethylenediaminetetraacetic acid (EDTA). In another preferredembodiment the restriction enzyme is inactivated by heat inactivatione.g. by incubation at 65° C.

In one embodiment, the plasmid DNA comprises at least one terminatorsequence. This sequence mediates transcriptional termination byproviding signals in the newly synthesized RNA that trigger processeswhich release the RNA from the transcriptional complex.

In a preferred embodiment, one or more steps of TFF are performed afterthe linearization reaction. The one or more steps of TFF may either beperformed as a diafiltration step for i) exchange the solvent of thelinearized DNA to conditions required for the transcription and/or forii) purifying the linearized DNA; and/or as a concentration step forconcentrating the linearized DNA. The conditioning may be performed byat least one step of diafiltration using TFF to a diafiltration solutionor buffer.

In a preferred embodiment of the method according to the presentinvention, the DNA provided in step A) is plasmid DNA as the DNAencoding the RNA and the method comprises subsequently to step A) theabove described steps:

A1) linearization of the plasmid DNA in a linearization reaction;

A2) optionally termination of the linearization reaction; and

A3) conditioning and/or purifying of the linearization reactioncomprising linearized plasmid DNA by one or more steps of TFF.

Step A3 according to the present invention comprises conditioning,and/or purifying of the linearization reaction comprising linearizedplasmid DNA by one or more steps of TFF. The at least one step of TFFmay comprise at least one diafiltration step using TFF and/or at leastone concentration step using TFF. The diafiltration and concentrationstep may be performed separately but they may also at least partiallyoverlap. The at least one or more steps of TFF may efficiently removecontaminants, such as HMWC and LMWC, e.g. DNA fragments, organicsolvents, buffer components such as salts and detergents. The use of TFFmay thus reduce or abolish the need to purify the DNA by means oforganic solvent extraction such as phenol/chloroform extraction and/oralcohol precipitation of nucleic acids such as RNA and DNA, e.g. by highsalt/alcohol precipitation such as NaCl/isopropanol precipitation.Further, it was found that, surprisingly, the use of TFF does notnegatively affect the stability of the DNA, e.g. due to shear stressduring pumping.

Thus, in a preferred embodiment, the method according to the presentinvention in step A3, particularly during conditioning, and/or purifyingof the linearization reaction does not comprise a step ofphenol/chloroform extraction and/or DNA/and/or RNA precipitation.

In a preferred embodiment, the at least one step of TFF in step A3comprises at least one concentration step. In this context it isparticularly preferred to concentrate the linearization reaction to atleast 90%, 80%, 70%, 60%, 50%, 40%, 30% or to at least 13% of theoriginal volume.

In a preferred embodiment, the at least one step of TFF in step A3comprises at least one concentration step, wherein the linearizedplasmid DNA is concentrated from an initial concentration of about 0.05g/l, 0.1 g/l, 0.15 g/l, 0.2 g/l, 0.25 g/l or 0.3 g/l linearized plasmidDNA to a final concentration of about 0.8 g/l, 0.9 g/l, 1.0 g/l, 1.1g/l, 1.2 g/l, 1.3 g/l, 1.4 g/l or about 1.5 g/l linearized plasmid DNA.

In another preferred embodiment, the at least one step of TFF in step A3comprises at least one concentration step, wherein the linearizationreaction is concentrated from 0.2 g/l DNA to 1.0 g/l or 1.5 g/l DNA.

In a further preferred embodiment, the at least one step of TFF in stepA3 comprises at least one diafiltration step.

In a preferred embodiment, the diafiltration step is performed withwater or an aqueous salt solution as diafiltration solution.Particularly preferred is a diafiltration step with water.

In a preferred embodiment, the at least one step of TFF in step A3 isperformed using from about 1 to about 20 diafiltration volumes (DV)diafiltration solution or buffer, preferably from about 1 to about 15 DVdiafiltration solution or buffer and more preferably from about 5 toabout 12 DV diafiltration solution or buffer and even more preferablyfrom about 6 to about 10 DV diafiltration solution or buffer. In aparticularly preferred embodiment, the at least one step of TFF isperformed using about 10 DV diafiltration solution or buffer,particularly water.

According to a particularly preferred embodiment, the at least one stepof TFF in step A3 comprises at least one concentration step and at leastone diafiltration step. Preferably, the at least one diafiltration stepis performed after the concentration step of step A3.

TFF may be carried out using any suitable filter membrane. For example,TFF may be carried out using a TFF hollow fibre membrane or a TFFmembrane cassette. The use of a TFF membrane cassette is preferred. Themolecular weight cutoff of the filter membrane may be selected dependingon the size of the DNA, particularly the plasmid DNA. The larger the DNAmolecule of interest, the higher the molecular weight cutoff of themembrane may be selected. In a preferred embodiment, the molecularweight cutoff of the filter membrane is 500 kDa, more preferably 200 kDaand most preferably 100 kDa. The filter membrane may comprise anysuitable filter material, e.g. polyethersulfone (PES), modifiedpolyethersulfone (mPES), polysulfone (PS), modified polysulfone (mPS),ceramics, polypropylene (PP), cellulose, regenerated cellulose or acellulose derivative e.g. cellulose acetate or combinations thereof.

Particularly preferred in this context is a cellulose-based membrane(cellulose, regenerated cellulose or a cellulose derivative) or a PES ormPES-based filter membrane, particularly with a MWCO of 100 kDa.

In a preferred embodiment, the DNA membrane load of the TFF membrane isabout 0.1 to about 10 mg/cm² and preferably from about 0.5 to about 2mg/cm².

In a preferred embodiment, the DNA membrane load of the TFF membrane isabout 0.1 to 0.6 mg/cm².

The feed flow rate in the at least one step of TFF in step A3 is 500 to1.500 l/h/m², preferably 600 to 1.200 l/h/m², more preferably 700 to1.000 l/h/m² and most preferably 750 to 900 l/h/m².

In a preferred embodiment, a TFF membrane cassette is used in step A3for the at least one TFF step. Surprisingly, it was found that TFFmembrane cassettes are particularly suitable for the method according tothe present invention. Examples of TFF membrane cassettes are SartoconSlice 200 100 kDa, PES (Sartorius), Sartcocon Slice 200 300 kDa, PES(Sartorius), Omega Centramate T OS300T02, PES 300 kDa (PALL), OmegaCentramate T OS100T02, PES 100 kDa (PALL) or NovaSet-LS ProStream (LowBinding mPES), 100 kDa (NovaSep). Another example is Sartocon Slice 200100 kDa, Hydrosart (Sartorius), which is a stabilised cellulose-basedmembrane, i.e. a cellulose derivative membrane.

Particularly preferred in this context is a TFF membrane cassettecomprising a cellulose-based membrane or a PES or mPES-based filtermembrane with a MWCO of 100 kDa.

Particularly preferred in this context is a TFF membrane cassettecomprising a mPES-based filter membrane with a MWCO of 100 kDa, e.g., acommercially available TFF membrane cassette such as NovaSep mPES with aMWCO of 100 kDa, or a cellulose-based membrane cassette with a MWCO of100 kDa, e.g. a commercially available TFF membrane cassette such asHydrosart (Sartorius).

Using TFF membrane cassettes provides the possibilities of higherpermeate flux rates compared to using hollow fibre membranes. A higherpermeate flux rate of the TFF step may result in a faster process timeand consequently lower production costs.

In a preferred embodiment of the present invention, the at least onestep of TFF in step A3 provides a permeate flux rate of at least 30l/h/m², 50 l/h/m², 70 l/h/m² or 90 l/h/m², preferably at least 100l/h/m², more preferably at least 110 l/h/m² and even more preferably atleast 120 l/h/m². Also preferably, the at least one step of TFF in stepA3 provides a permeate flux rate of 30 l/h/m² to 100 l/h/m².

In another preferred embodiment, the transmembrane pressure (TMP) overthe TFF membrane cassette in this step is from about 0.01 (0.1 bar) toabout 0.3 MPa (3 bar) and preferably from about 0.1 (1 bar) to about 0.2MPa (2 bar) and most preferably is 0.1 MPa (1 bar). Furthermore, adeltaP (dp) of from about 0.05 (0.5 bar) to about 0.5 MPa (5 bar) andparticularly of about 0.1 MPa (1 bar) is preferred. The values for TMPand dp of about 0.1 MPa (1 bar) and about 0.1 MPa (1 bar), respectively,are particularly preferred because under these conditions the process isnot cake layer driven.

In a preferred embodiment, at least one or more steps of TFF in step A3comprise using a TFF membrane cassette comprising a cellulose orcellulose derivative-based membrane. In a preferred embodiment, both theconcentration and the diafiltration step of TFF in step A3 comprisesusing a TFF membrane cassette comprising a cellulose or cellulosederivative-based membrane. A Hydrosart membrane (Sartorius), whichprovides high permeate flux rates and at the same time a high stabilityin the presence of organic solvents such as acetonitrile, isparticularly preferred.

In another preferred embodiment, at least one or more steps of TFF instep A3 comprise using a TFF PES- or mPES-based membrane cassette, e.g.a PES-based membrane from Sartorius, more preferably a mPES-basedmembrane cassette from NovaSep.

In yet another preferred embodiment, the at least one or more steps ofTFF in step A3 comprises using a TFF membrane with a molecular weightcutoff of about 100 kDa.

In yet another preferred embodiment, the at least one or more steps ofTFF in step A3 comprises using a TFF membrane with a molecular weightcutoff of about 300 kDa.

Step B:

In step B of the method according to the present invention the DNA(template) may either be transcribed in vivo or in vitro to yield asolution comprising RNA.

In a preferred embodiment, the DNA template is transcribed in vitro toyield a solution comprising RNA in a process called DNA dependent RNA invitro transcription. The DNA dependent RNA in vitro transcription may beperformed in the presence of an in vitro transcription mix (IVT-mix).The IVT-mix is a complex mixture of several high and low molecularweight compounds (HMWC/LMWC), e.g. linear template DNA, nucleosidetriphosphates (NTPs), proteins, e.g. proteins of the transcriptionmachinery and/or enzymes, spermidine and salts.

In general, RNA can be produced “in vitro” from a PCR-based DNAtemplate, or a plasmid DNA-based linearized DNA template using DNAdependent RNA polymerases.

Particularly preferred is DNA dependent RNA in vitro transcription usinga linearized plasmid DNA template.

The template DNA, preferably the linearized template DNA plasmid istranscribed into RNA using DNA dependent RNA in vitro transcription.That reaction typically comprises a transcription buffer, nucleotidetriphosphates (NTPs), an RNase inhibitor and a DNA-dependent RNApolymerase. The NTPs can be selected from, but are not limited to thosedescribed herein including naturally occurring and non-naturallyoccurring (modified) NTPs. The DNA-dependent RNA polymerase can beselected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase, SP6 RNA polymerase and mutant polymerases such as, but notlimited to, polymerases able to incorporate modified NTPs.

In preferred embodiments, viral DNA-dependent RNA polymerases is used asthe RNA polymerase. More preferably, a bacteriophage DNA-dependent RNApolymerase selected from the group comprising T3, T7 and/or Sp6polymerases is used as the RNA polymerase Most preferably T7 RNApolymerase is used as an enzyme for DNA-dependent RNA in vitrotranscription.

In a preferred embodiment, 1-1000 Units (U DNA-dependent RNA polymeraseper μg DNA template may be used. Even more preferred is a concentrationof 100 U/DNA-dependent RNA polymerase per μg DNA template

During RNA polymerization, the RNA may be co-transcriptionally capped atthe 5′ end with a cap analogue as defined herein (e.g. N7-MeGpppG).

As transcription buffer following buffers are preferred: 40 mM Tris pH7.5 or 80 mM HEPES. 80 mM HEPES is particularly preferred.

In another preferred embodiment, 40 mM Tris-HCl buffer pH 7.5 ispreferred.

In a preferred embodiment, a template DNA concentration of from about 10to about 500 μg/ml is used. Particularly preferred is a template DNAconcentration of about 50 μg/ml.

For the transcription, nucleotide triphosphates of the desired chemistryare used, including naturally occurring nucleotides (e.g. at least oneof the nucleotides ATP, CTP, UTP and GTP) and/or modified nucleotides,preferably modified nucleotides as described herein, or any combinationthereof.

ATP, CTP, UTP and GTP are preferably used in a concentration of 0.5-10mM, preferably in a concentration of 3-5 mM and most preferably in aconcentration of 4 mM.

Useful cap analogs include, but are not limited to, N7-MeGpppG(=m7G(5′)ppp(5′)G), m7G(5′)ppp(5′)A, ARCA (anti-reverse CAP analogue,modified ARCA (e.g. phosphothioate modified ARCA), inosine,N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine. If 5′-CAP (cap analog) is used, the concentration ofGTP is decreased compared to the other used nucleotides. Preferably 10to 50% of GTP is used compared to the concentration of ATP, CTP and UTP.Most preferably 20-30% of GTP is used.

Furthermore, the cap analog is preferably used in a concentration whichis at least the same as the concentration of ATP, CTP and UTP.

The ratio of cap analog:nucleotide and preferably cap analog:GTP can bevaried from 10:1 to 1:1 to balance the percentage of capped productswith the efficiency of the transcription reaction, preferably a ratio ofcap analog:GTP of 4:1-5:1 is used. In this context, it is particularlypreferred to use 5.8 mM Cap analog and 1.45 mM GTP if ATP, UTP and CTPare used in a concentration of 4 mM.

MgCl₂ can optionally be added to the transcription reaction. Preferredis a concentration of 1-100 mM. Particularly preferred is aconcentration of 5-30 mM and most preferably 24 mM MgCl₂ is used.

Dithiothreitol (DTT) can optionally be added to the transcriptionreaction, preferably at a concentration of 1-100 mM, more preferably10-100 mM, and most preferably 40 mM.

An RNase inhibitor can optionally be added to the transcriptionreaction, preferably 0.1-1 U/μl, most preferably 0.2 U/μl.

E. coli pyrophosphatase can optionally be added to the transcriptionreaction, preferably in a concentration of 1-10 U/μg template DNA, andmore preferably in a concentration of 5 U/μg template DNA. This ensuresthat magnesium, which is essential for transcription, remains insolution and does not precipitate as magnesium pyrophosphate.

Bovine serum albumine (BSA) can optionally be used in the transcriptionreaction, preferably in a concentration of 1-1000 μg/ml, more preferablyin a concentration of 100 μg/ml. Most preferably, BSA is not present inthe transcription reaction.

In a particularly preferred embodiment, the IVT-mix comprisespolycationic aliphatic amines, preferably spermidine. The polycationicaliphatic amines may interact with the negatively charged nucleic acids.The presence of the polycationic aliphatic amine, preferably spermidine,is known to assist the RNA in vitro transcription process. However,residual spermidine has to be depleted from the RNA solution,particularly if the RNA is used for therapeutic purposes.

In the light of the above, there is therefore a need to removespermidine from the solution of the in vitro transcribed RNA insubsequent purification steps.

In a preferred embodiment, the IVT-mix comprises from about 0.1 mM toabout 10 mM spermidine, preferably from about 1 mM to about 5 mM, andmost preferably about 2 mM spermidine.

In a particularly preferred embodiment, the RNA in vitro transcriptionreaction comprises the following components:

1 μg linearized plasmid DNA,

4 mM ATP, CTP and UTP,

1.45 mM GTP,

5.8 mM CAP analogue,

80 mM HEPES,

24 mM MgCl₂,

2 mM Spermidine,

40 mM DTT,

5 U pyrophosphatase,

4 U RNase inhibitor, and

100 U T7 RNA polymerase.

The in vitro transcription reaction may be incubated at 37° C.preferably for at least 4 hours.

In another particularly preferred embodiment, the RNA in vitrotranscription for large-scale reactions comprises the followingcomponents:

25 mg linearized plasmid DNA,

20 mM ATP, CTP and UTP,

7.25 mM GTP,

29 mM CAP analogue,

80 mM HEPES,

24 mM MgCl₂,

2 mM Spermidine,

40 mM DTT, and

the enzymes pyrophosphatase (5 U per μg DNA), RNAse inhibitor (0.2 U/μl)and T7 RNA Polymerase (100 U per μg DNA).

The RNA in vitro transcription reaction may be incubated at 37° C.,preferably for at least 4 hours.

After RNA in vitro transcription of the DNA, the transcribed RNA ispresent in a solution. The solution typically comprises components ofthe IVT-mix, which typically still comprise proteins such as polymeraseand other enzymes, BSA, HEPES, pyrophosphatase etc, nucleotides, saltsand spermidine.

Step C:

Step C according to the present invention comprises conditioning and/orpurifying of the solution comprising transcribed RNA by one or moresteps of TFF. The at least one step of TFF may comprise at least onediafiltration step and/or at least one concentration step. Thediafiltration and concentration steps may be performed separately, butthey may also at least partially overlap. The at least one or more stepsof TFF may efficiently remove contaminants, such as HMWC and LMWC, e.g.RNA fragments; DNA fragments, proteins, organic solvents, nucleosidetriphosphates, spermidine and buffer components such as salts anddetergents. The use of TFF may thus reduce or abolish the need to purifythe RNA by means of organic solvent extraction such as phenol/chloroformextraction and/or alcohol precipitation of nucleic acids such as RNA andDNA, e.g. by high salt/alcohol precipitation such as NaCl/isopropanolprecipitation. Further, it was found that, surprisingly, the use of TFFdoes not negatively affect the stability of the RNA, e.g. due to shearstress during pumping.

Thus, in a preferred embodiment, the method according to the presentinvention does not comprise a step of phenol/chloroform extractionand/or DNA and/or RNA precipitation. It is one advantage of the methodaccording to the present invention that the purified RNA may provideincreased storage stability after at least one step of TFF compared e.g.to the storage stability of the RNA in the IVT-mix.

Further, the method according to the present invention does not comprisethe addition of a protein denaturing agent such as urea, guanidiniumthiocyanate, KCl, sodium dodecyl sulfate, sarcosyl or other detergentsto the RNA in vitro transcription reaction mixture, before it issubjected to tangential flow filtration.

In a preferred embodiment, the at least one step of TFF in step Ccomprises at least one diafiltration step.

The TFF step in step C2 is preferably a diafiltration step which ispreferably performed with water.

In a preferred embodiment, the at least one step of TFF in step C2 isperformed using from about 1 to about 20 diafiltration volumes (DV)diafiltration solution or buffer, preferably from about 1 to about 15 DVdiafiltration solution or buffer and more preferably from about 5 toabout 12 DV diafiltration solution or buffer and even more preferablyfrom about 5 to about 10 DV diafiltration solution or buffer. In aparticularly preferred embodiment, the at least one step of TFF isperformed using about 10 DV diafiltration solution or buffer.

In a preferred embodiment, the diafiltration step is performed withwater or an aqueous salt solution as diafiltration solution or buffer.In a preferred embodiment, the salt of the aqueous salt solution maycomprise alkaline metal halides such as NaCl, LiCl or KCl; organic saltssuch as NaOAc, earth alkaline metal halides such as CaCl₂); alkalinemetal phosphates such as Na₃PO₄, Na₂HPO₄, NaH₂PO₄; or combinationsthereof.

In a preferred embodiment, the salt of the aqueous salt solutioncomprises alkaline metal halides such as NaCl, LiCl or KCl; earthalkaline metal halides such as CaCl₂) or combinations thereof.

In a more preferred embodiment, the aqueous salt solution comprises fromabout 0.1 M alkaline metal halide to about 1 M alkaline metal halide,more preferably from about 0.2 to about 0.5 M alkaline metal halide.Concentrations higher than the preferred range may lead to RNAprecipitation and, consequently, blocking of the TFF membrane.

In another preferred embodiment, the aqueous salt solution comprisesNaCl. In a more preferred embodiment, the aqueous salt solutioncomprises from about 0.1 M NaCl to about 1 M NaCl, more preferably fromabout 0.2 to about 0.5 M NaCl.

In another preferred embodiment, the diafiltration solution does notcomprise buffering salts.

In another preferred embodiment, the diafiltration solution is water,preferably distilled and sterile water, more preferably water forinjection.

In a preferred embodiment, the at least one step of TFF is performedusing from about 1 to about 20 diafiltration volumes (DV) diafiltrationsolution or buffer, preferably from about 1 to about 15 DV diafiltrationsolution or buffer and more preferably from about 5 to about 12 DVdiafiltration solution or buffer and even more preferably from about 5to about 10 DV diafiltration solution or buffer. In a particularlypreferred embodiment, the at least one step of TFF is performed usingabout 10 DV diafiltration solution or buffer.

All TFF steps in step C may be carried out using any suitable filtermembrane. For example, TFF may be carried out using a TFF hollow fibremembrane or a TFF membrane cassette. The use of a TFF membrane cassetteis preferred. The molecular weight cutoff of the filter membrane may beselected depending on the size of the produced desired RNA molecules.The larger the RNA molecule of interest, the higher the molecular weightcutoff of the membrane may be selected, respectively. In a preferredembodiment, the molecular weight cutoff of the filter membrane is ≤500kDa, more preferably 200 kDa and most preferably 100 kDa. The filtermembrane may comprise any suitable filter material, e.g.polyethersulfone (PES), modified polyethersulfone (mPES), polysulfone(PS), modified polysulfone (mPS), ceramics, polypropylene (PP),cellulose, regenerated cellulose or a cellulose derivative e.g.cellulose acetate or combinations thereof. Particularly preferred inthis context is a cellulose-based membrane (cellulose, regeneratedcellulose or a cellulose derivative), a PES or mPES-based filtermembrane, particularly with a MWCO of 100 kDa.

In a preferred embodiment, the RNA membrane load of the TFF membrane isabout 1 to about 10 mg/cm² and preferably from about 2 to about 6mg/cm².

In a particularly preferred embodiment, the RNA membrane load of the TFFmembrane in step C2 is about 2.5 to about 6.5 mg/cm².

The feed flow rate in the at least one step of TFF in step C2 is 100 to1.500 l/h/m², preferably 150 to 1.300 l/h/m², more preferably 200 to1.100 l/h/m² and most preferably 300 to 1.050 l/h/m².

In a preferred embodiment, a TFF membrane cassette is used.Surprisingly, it was found that TFF membrane cassettes are particularlysuitable for the method according to the present invention. Examples ofTFF membrane cassettes are Sartocon Slice 200 100 kDa, PES (Sartorius),Sartcocon Slice 200 300 kDa, PES (Sartorius), Omega Centramate TOS300T02, PES 300 kDa (PALL), Omega Centramate T OS100T02, PES 100 kDa(PALL) or NovaSet-LS ProStream (Low Binding mPES), 100 kDa (NovaSep).Another example is Sartocon Slice 200 100 kDa, Hydrosart (Sartorius),which is a stabilised cellulose-based membrane, i.e. a cellulosederivative membrane.

Particularly preferred in this context is a TFF membrane cassettecomprising an mPES-based filter membrane with a MWCO of 100 kDa, e.g., acommercially available TFF membrane cassette such as NovaSep mPES with aMWCO of 100 kDa, or a cellulose-based membrane cassette with a MWCO of100 kDa, e.g. a commercially available TFF membrane cassette such asHydrosart (Sartorius).

Using TFF membrane cassettes provides the possibilities of higherpermeate flux rates compared to using hollow fibre membranes. A higherpermeate flux rate of the TFF step may result in a higher concentrationof the retentate and thus a more concentrated RNA product and a fasterprocess time and consequently lower production costs.

In a preferred embodiment of the present invention, the at least onestep of TFF in step C2 provides a permeate flux rate of at least 20l/h/m², 40 l/h/m², 60 l/h/m², 80 l/h/m² or 90 l/h/m², preferably atleast 100 l/h/m², more preferably at least 110 l/h/m² and even morepreferably at least 120 l/h/m². Also preferably, the at least one stepof TFF in step A3 provides a permeate flux rate of 20 l/h/m² to 100l/h/m².

In another preferred embodiment, the transmembrane pressure (TMP) overthe TFF membrane cassette in step C2 is from about 0.01 (0.1 bar) toabout 0.3 MPa (3 bar) and preferably from about 0.05 (0.5 bar) to about0.2 MPa (2 bar) and most preferably from about 0.075 (0.75 bar) to about0.15 MPa (1.5 bar) or from about 0.1 MPa (1 bar) to about 0.15 MPa (1.5bar). Furthermore, a deltaP (dp) of from about 0.05 (0.5 bar) to about0.5 MPa (5 bar), more preferably of from about 0.05 (0.5 bar) to about0.1 MPa (1 bar) and particularly of about 0.1 MPa (1 bar) is preferred.The values for TMP and dp of about 0.15 MPa (1.5 bar) and about 0.1 Mpa(1 bar), respectively, are particularly preferred because under theseconditions the process is not cake layer driven.

In a preferred embodiment, the values for TMP are from about 0.1 toabout 0.15 and dp are about 0.1 MPa.

In a preferred embodiment, at least one or more steps of TFF compriseusing a TFF membrane cassette comprising a cellulose-based membrane. AHydrosart membrane (Sartorius), and a NovaSep membrane which providehigh permeate flux rates and at the same time a high stability in thepresence of organic solvents such as acetonitrile, is particularlypreferred.

In another preferred embodiment, at least one or more steps of TFFcomprise using a TFF PES- or mPES-based membrane cassette e.g. aPES-based membrane more preferably an mPES-based membrane cassette fromNovaSep.

In yet another preferred embodiment, the at least one or more steps ofTFF comprises using a TFF membrane with a molecular weight cutoff ofabout 100 kDa.

In yet another preferred embodiment, the at least one or more steps ofTFF comprises using a TFF membrane with a molecular weight cutoff ofabout 50 kDa.

In one embodiment of the inventive method, the same TFF membranecassette is used for more than one or even all TFF steps. The use of thesame TFF membrane for more than one TFF steps is particularlyadvantageous because it reduces the amount of disposable waste, costsand time of the inventive RNA production method.

In one preferred embodiment, the method according to the presentinvention does not comprise using a TFF hollow fibre membrane in any ofthe TFF steps.

In yet another preferred embodiment, the step C) of the inventive methodcomprises at least one further purification method C3 before or afterthe one or more steps of TFF. In a preferred embodiment, the furtherpurification method is performed after a first TFF step C2 and before asecond TFF step C4. In a preferred embodiment, the at least one furtherpurification method is selected from the group consisting of cationexchange chromatography, anion exchange chromatography, membraneabsorbers, reversed phase chromatography, normal phase chromatography,size exclusion chromatography, hydrophobic interaction chromatography,mixed mode chromatography, affinity chromatography, hydroxylapatite (HA)chromatography, or combinations thereof. In another embodiment the atleast one further purification method does not comprise any ofhydroxyapatite chromatography and core bead flow-through chromatography.

In a preferred embodiment, the at least one further purification methodis performed before the at least one step of TFF.

In a further preferred embodiment the at least one further purificationmethod is performed by means of high performance liquid chromatography(HPLC) or low normal pressure liquid chromatography methods. A HPLCmethod is particularly preferred.

In another preferred embodiment, the at least one further purificationmethod is a reversed phase chromatography method, preferably a reversedphase HPLC (RP-HPLC) method. Preferably, the reversed phasechromatography comprises using a porous reserved phase as stationaryphase.

In a preferred embodiment of the method according to the invention, theporous reversed phase material is provided with a particle size of 8.0μm to 50 μm, in particular 8.0 to 30 μm, still more preferably about 30μm. The reversed phase material may be present in the form of smallspheres. The method according to the invention may be performedparticularly favourably with a porous reversed phase with this particlesize, optionally in bead form, wherein particularly good separationresults are obtained.

In another preferred embodiment, the reversed phase used in the methodaccording to the invention may be porous and may have specific particlesizes. With stationary reversed phases which are not porous and thusdiffer completely with regard to particle size from the subject matterof the present invention as described for example by A. Azarani and K.H. Hecker (Nucleic Acids Research, vol. 29, no. 2 e7), on the otherhand, excessively high pressures are built up, such that preparativepurification of the RNA is possible only with great difficulty, if atall.

In a preferred embodiment of the method according to the invention, thereversed phase has a pore size of 1000 Å to 5000 Å, in particular a poresize of 1000 Å to 4000 Å, more preferably 1500 Å to 4000 Å, 2000 Å to4000 Å or 2500 Å to 4000 Å. Particularly preferred pore sizes for thereversed phases are 1000 Å to 2000 Å, more preferably 1000 Å to 1500 Åand most preferably 1000 to 1200 Å or 3500-4500 Å. Most preferred is apore size of 4000 Å. With a reversed phase having these pore sizes,particularly good results are achieved with regard to purification ofthe RNA using the method according to the invention, in particular theelevated pressures built up in the method according to A. Azarani and K.H. Hecker are thus avoided, whereby preparative separation is madepossible in a particularly favourable manner. At pore sizes of below1000 Å separation of RNA molecules becomes poorer.

A pore size of 1000 Å to 5000 Å, in particular a pore size of 1000 Å to4000 Å, more preferably 1500 Å to 4000 Å, 2000 Å to 4000 Å or 2500 Å to4000 Å may be suitable to separate a RNA from other components of amixture, the RNA having a size as mentioned above of up to about 15000nucleotides (as single stranded RNA molecule) or base pairs (as doublestranded RNA molecule), in particular 100 to 10000, more preferably 500to 10000 nucleotides or base pairs, even more preferably 800 to 5000nucleotides or base pairs and even more preferably 800 to 2000nucleotides or base pairs. However, the pore size of the reversed phasemay also be selected in dependence of the size of the RNA to beseparated, i.e. a larger pore size may be selected, if larger RNAmolecules are to be separated and smaller pore sizes may be selected, ifsmaller RNA molecules may be selected. This is due to the effect thatthe retention of the RNA molecules and the separation not only dependson the interaction of the (reversed) phase but also on the possibilityof molecules to get inside the pores of the matrix and thus provide afurther retention effect. Without being limited thereto, e.g. a poresize for the reversed phase of about 2000 Å to about 5000 Å, morepreferably of about 2500 to about 4000, most preferably of about 3500 toabout 4500 Å, may thus be used to separate larger RNA molecules, e.g.RNA molecules of 100 to 10000, more preferably 500 to 10000 nucleotidesor base pairs, even more preferably 800 to 5000 nucleotides or basepairs and even more preferably 800 to 2000 nucleotides or base pairs.Alternatively, without being limited thereto, a pore size for thereversed phases of about 1000 to about 2500 Å, more preferably of about1000 Å to about 2000 Å, and most preferably of about 1000 Å to 1200 Åmay be used to separate smaller RNA molecules, e.g. RNA molecules ofabout 30-1000, 50-1000 or 100-1000 or 20-200, 20-100, 20-50 or 20-30nucleotides may also be separated in this way.

In general, any material known to be used as reverse phase stationaryphase, in particular any polymeric material may be used for theinventive method, if that material can be provided in porous form. Thestationary phase may be composed of organic and/or inorganic material.Examples for polymers to be used for the present invention are(non-alkylated) polystyrenes, (non-alkylated)polystyrenedivinylbenzenes, monolithic materials, silica gel, silica gelmodified with non-polar residues, particularly silica gel modified withalkyl containing residues, more preferably with butyl-, octyl and/oroctadecyl containing residues, silica gel modified with phenylicresidues, polymethacrylates, etc. or other materials suitable e.g. forgel chromatography or other chromatographic methods as mentioned above,such as dextran, including e.g. Sephadex® and Sephacryl® materials,agarose, dextran/agarose mixtures, polyacrylamide, etc.

In a particularly preferred embodiment, the material for the reversedphase is a porous polystyrene polymer, a (non-alkylated) (porous)polystyrenedivinylbenzene polymer, porous silica gel, porous silica gelmodified with non-polar residues, particularly porous silica gelmodified with alkyl containing residues, more preferably with butyl-,octyl and/or octadecyl containing residues, porous silica gel modifiedwith phenylic residues, porous polymethacrylates, wherein in particulara porous polystyrene polymer or a non-alkylated (porous)polystyrenedivinylbenzene may be used. Stationary phases withpolystyrenedivinylbenzene are known per se. The per se knownpolystyrenedivinyl-benzenes already used for HPLC methods, which arecommercially obtainable, may be used for the method according to theinvention.

A non-alkylated porous polystyrenedivinylbenzene which is veryparticularly preferred for the method according to the invention is onewhich, without being limited thereto, may have in particular a particlesize of 8.0±1.5 μm, in particular 8.0±0.5 μm, and a pore size of1000-1500 Å, in particular 1000-1200 Å or 3500-4500 Å and mostpreferably a particle size of 4000 Å. With this material for thereversed phases, the above-described advantages of the method accordingto the invention may be achieved in a particularly favourable manner.

This stationary phase described in greater detail above isconventionally located in a column. V2A steel is conventionally used asthe material for the column, but other materials may also be used forthe column provided they are suitable for the conditions prevailingduring HPLC. Conventionally the column is straight. It is favourable forthe HPLC column to have a length of 5 cm to 100 cm and a diameter of 4mm to 50 cm. Columns used for the method according to the invention mayin particular have the following dimensions: 25 cm long and 20 mm indiameter or 25 cm long and 50 mm in diameter, or 25 cm long and 10 cm indiameter or any other dimension with regard to length and diameter,which is suitable for preparative recovery of RNA, even lengths ofseveral metres and also larger diameters being feasible in the case ofupscaling. The dimensions are here geared towards what is technicallypossible with liquid chromatography.

Selection of the mobile phase depends on the type of separation desired.This means that the mobile phase established for a specific separation,as may be known for example from the prior art, cannot bestraightforwardly applied to a different separation problem with asufficient prospect of success. For each separation problem, the idealelution conditions, in particular the mobile phase used, have to bedetermined by empirical testing.

In a preferred embodiment of the HPLC method according to the invention,a mixture of an aqueous solvent and an organic solvent is used as themobile phase for eluting the RNA. It is favourable for a buffer to beused as the aqueous solvent which has in particular a pH of 6.0-8.0, forexample of about 7, for example. 7.0; preferably the buffer istriethylammonium acetate (TEAA), particularly preferably a 0.02 M to 0.5M, in particular 0.08 M to 0.12 M, very particularly an about 0.1 M TEAAbuffer, which, as described above, also acts as a counterion to the RNAin the ion pair method.

In a preferred embodiment, the organic solvent which is used in themobile phase comprises acetonitrile, methanol, ethanol, 1-propanol,2-propanol and acetone or a mixture thereof, very particularlypreferably acetonitrile. With these organic solvents, in particularacetonitrile, purification of the RNA proceeds in a particularlyfavourable manner with the method according to the invention.

In a particularly preferred embodiment of the method according to theinvention, the mobile phase is a mixture of 0.1 M triethylammoniumacetate, pH 7, and acetonitrile.

It has proven particularly favourable for the method according to theinvention for the mobile phase to contain 5.0 vol. % to 25.0 vol. %organic solvent, relative to the mobile phase, and for this to be madeup to 100 vol. % with the aqueous solvent. Typically, in the event ofgradient separation, the proportion of organic solvent is increased, inparticular by at least 10%, more preferably by at least 50% and mostpreferably by at least 100%, optionally by at least 200%, relative tothe initial vol. % in the mobile phase. In a preferred embodiment, inthe method according to the invention the proportion of organic solventin the mobile phase amounts in the course of HPLC separation to 3 to 9,preferably 4 to 7.5, in particular 5.0 vol. %, in each case relative tothe mobile phase. More preferably, the proportion of organic solvent inthe mobile phase is increased in the course of HPLC separation from 3 to9, in particular 5.0 vol. % to up to 20.0 vol. %, in each case relativeto the mobile phase. Still more preferably, the method is performed insuch a way that the proportion of organic solvent in the mobile phase isincreased in the course of HPLC separation from 6.5 to 8.5, inparticular 7.5 vol. %, to up to 17.5 vol. %, in each case relative tothe mobile phase.

It has proven even more particularly favourable for the method accordingto the invention for the mobile phase to contain 7.5 vol. % to 17.5 vol.% organic solvent, relative to the mobile phase, and for this to be madeup to 100 vol. % with the aqueous buffered solvent.

In the case of the method according to the invention elution may proceedisocratically or by means of gradient separation. In isocraticseparation, elution of the RNA proceeds with a single eluent or aconstant mixture of a plurality of eluents, wherein the solventsdescribed above in detail may be used as eluent.

In a preferred embodiment of the method according to the invention,gradient separation is performed. In this respect, the composition ofthe eluent is varied by means of a gradient program. The equipmentnecessary for gradient separation is known to a person skilled in theart. Gradient elution may here proceed either on the low pressure sideby mixing chambers or on the high pressure side by further pumps.

Preferably, in the method according to the invention, the proportion oforganic solvent, as described above, is increased relative to theaqueous solvent during gradient separation. The above-described agentsmay here be used as the aqueous solvent and the likewise above-describedagents may be used as the organic solvent.

For example, the proportion of organic solvent in the mobile phase maybe increased in the course of HPLC separation from 5.0 vol. % to 20.0vol. %, in each case relative to the mobile phase. In particular, theproportion of organic solvent in the mobile phase may be increased inthe course of HPLC separation from 7.5 vol. % to 17.5 vol. %, inparticular 9.5 to 14.5 vol. %, in each case relative to the mobilephase.

The following gradient program has proven particularly favourable forthe purification of RNA with the method according to the invention:

-   -   Eluent A: 0.1 M triethylammonium acetate, pH 7    -   Eluent B: 0.1 M triethylammonium acetate, pH 7, with 25 vol. %        acetonitrile    -   Eluent composition:        -   start: 62% A and 38% B (1st to 3rd minute)        -   increase to 58% B (1.67% increase in B per minute),            (3rd-15th minute) 100% B (15th to 20th minute)

Another example of a gradient program is described below, the sameeluent A and B being used:

-   -   Eluent composition:        -   starting level: 62% A and 38% B (1st-3rd min)        -   separation range I: gradient 38%-49.5% B (5.75% increase in            B/min) (3rd-5th min)        -   separation range II: gradient 49.5%-57% B (0.83% increase in            B/min) (5th-14th min)        -   rinsing range: 100% B (15th-20th min)

The flow rate of the eluent is so selected that good separation of theRNA from the other constituents contained in the sample to beinvestigated takes place. The eluent flow rate selected for the methodaccording to the invention may amount to from 1 ml/min to several litresper minute (in the case of upscaling), in particular about 1 to 1000ml/min, more preferably 5 ml to 500 ml/min, even more preferably morethan 100 ml/min, depending on the type and scope of the upscaling. Thisflow rate may be established and regulated by the pump.

Detection proceeds favourably with a UV detector at 254 nm, wherein areference measurement may be made at 600 nm. However, any otherdetection method may alternatively be used, with which the RNA describedabove in greater detail may be detected in satisfactory and reliablemanner.

In preferred embodiments, the RP-HPLC is performed as described in WO2008/077592.

As described above, the use of reversed phase chromatography methodstypically requires the use of organic solvents such as acetonitrile(ACN), methanol, ethanol, 1-propanol, 2-propanol, trifluoroacetic acid(TFA), trifluoroethanol (TFE) or combinations thereof. However, theseorganic solvents may need to be removed from the RNA-containing poolafterwards. Furthermore, other contaminations derived from priorproduction or purification steps, such as spermidine, may still bepresent in the RNA-containing pool after RP-HPLC and need to be removed.

In a preferred embodiment, the at least one step of TFF in step C, maybe performed after performing the at least one further purificationmethod. This at least one step of TFF after the optional at least onepurification method may comprise at least a first step of diafiltration.Preferably, the first diafiltration step is performed with an aqueoussalt solution as diafiltration solution. In a preferred embodiment, thesalt of the aqueous salt solution may comprise alkaline metal halidessuch as NaCl, LiCl or KCl; organic salts such as NaOAc, earth alkalinemetal halides such as CaCl₂; alkaline metal phosphates such as Na₃PO₄,Na₂HPO₄, NaH₂PO₄; or combinations thereof. In a preferred embodiment,the salt of the aqueous salt solution may comprise alkaline metalhalides such as NaCl, LiCl or KCl; earth alkaline metal halides such asCaCl₂). In a more preferred embodiment, the aqueous salt solutioncomprises from about 0.1 M alkaline metal halide to about 1 M alkalinemetal halide, more preferably from about 0.2 to about 0.5 M alkalinemetal halide. In another preferred embodiment, the aqueous salt solutioncomprises NaCl. In a more preferred embodiment, the aqueous saltsolution comprises about 0.1 M NaCl to about 1 M NaCl, more preferablyfrom about 0.2 to about 0.5 M NaCl. In a particularly preferredembodiment, the aqueous salt solution comprises 0.2 M NaCl. In anotherpreferred embodiment, the aqueous salt solution does not comprisebuffering salts. The presence of the salt may be advantageous forremoving contaminating spermidine from the RNA-Pool. In a preferredembodiment, the first diafiltration step is performed using from about 1to about 20 DV diafiltration solution, preferably from about 1 to about15 DV diafiltration solution and more preferably from about 5 to about12 DV diafiltration solution and even more preferably from about 7 toabout 10 DV diafiltration solution. In a particularly preferredembodiment, the first diafiltration step is performed using about 10 DVdiafiltration solution.

In a preferred embodiment, the RNA membrane load in the at least onestep of TFF membrane (after the optional at least one purificationmethod) is about 1 to about 10 mg/cm² and preferably from about 1 toabout 5 mg/cm².

In a particularly preferred embodiment, the RNA membrane load of the TFFmembrane is about 2 mg/cm² to about 2.5 mg/cm².

In a preferred embodiment of the present invention, the at least onestep of TFF (after the optional at least one purification method)provides a feed flow rate of 500 to 2.000 l/h/m², preferably of 600 to1.800 l/h/m², more preferably of 700 to 1.600 l/h/m² and most preferablyof 900 to 1.500 l/h/m².

In a preferred embodiment of the present invention, the at least onestep of TFF (after the optional at least one purification method)provides a permeate flux rate of at least 20 l/h/m², preferably at least501/h/m², more preferably at least 100 l/h/m² and even more preferablyat least 150 l/h/m².

In a preferred embodiment of the present invention, the at least onestep of TFF (after the optional at least one purification method)provides a permeate flux rate of about 25 l/h/m² to about 140 l/h/m².

In another preferred embodiment, the transmembrane pressure (TMP) overthe TFF membrane cassette in the at least one step of TFF (after theoptional at least one purification method) is from about 0.01 (0.1 bar)to about 0.3 MPa (3 bar) and preferably from about 0.05 (0.5 bar) toabout 0.2 MPa (2 bar) and most preferably from about 0.075 (0.75 bar) toabout 0.15 MPa (1.5 bar). Furthermore, a deltaP (dp) of from about 0.05(0.5 bar) to about 0.5 MPa (5 bar), more preferably of from about 0.05(0.5 bar) to about 0.1 MPa (1 bar) and particularly of about 0.1 MPa (1bar) is preferred. The values for TMP and dp of about 0.15 MPa (1.5 bar)and about 0.1 MPa (1 bar), respectively, are particularly preferredbecause under these conditions the process is not cake layer driven.

In a preferred embodiment, the values for TMP are from about 0.1 (1 bar)to about 0.15 (1.5 bar) and dp are about 0.1 MPa (1 bar).

In another preferred embodiment, the at least one step of TFF after theoptional at least one further purification method comprises at least onestep of concentrating the RNA, which is preferably performed before thefirst diafiltration step described above. In a preferred embodiment theRNA-Pool resulting from the optional at least one further purificationmethod is concentrated to a concentration of from about 0.1 g/l to about10 g/l, preferably to a concentration of from about 1 g/l to about 10g/l and more preferably to a concentration of from about 2 g/l to about5 g/l. In a particularly preferred embodiment, the RNA-Pool resultingfrom the optional at least one further purification method isconcentrated to a concentration of from about 0.1 g/l to about 5 g/l.The concentrating of RNA by TFF may reduce the overall process time.

In yet another preferred embodiment, the first diafiltration step afterthe optional at least one purification method is followed by a seconddiafiltration step using TFF. In an even more preferred embodiment thesecond diafiltration step is performed using water as diafiltrationsolution. In a preferred embodiment, the first diafiltration step isperformed using from about 1 to about 20 DV diafiltration solution,preferably from about 1 to about 15 DV diafiltration solution and morepreferably from about 5 to about 12 DV diafiltration solution and evenmore preferably from about 6 to about 10 DV diafiltration solution. In aparticularly preferred embodiment, the second diafiltration step isperformed using about 10 DV diafiltration solution.

In a preferred embodiment, a step of concentrating the RNA using TFF isperformed after the second diafiltration step.

In a particularly preferred embodiment, the at least one furtherpurification method is followed by at least one step of concentratingthe RNA, at least one first diafiltration step and at least one seconddiafiltration step using TFF as described above.

In a particularly preferred embodiment, the diafiltration step after theoptional at least one further purification method as well as theconcentration step after the optional at least one further purificationmethod using TFF are performed at temperatures from 0° C. to 20° C.,more preferably from 5° C. to 20° C., even more preferably from 10° C.to 20° C., even more preferably at temperatures below 20° C., even morepreferably at temperatures below 17° C.

In yet another particularly preferred embodiment, the method comprisesin step C) the steps of:

C1) optionally termination of transcription;

C2) conditioning and/or purifying of the solution comprising the invitro transcribed RNA by one or more steps of TFF;

C3) purifying the RNA by any further purification method as describedabove, preferably by using a method selected from the group consistingof cation exchange chromatography, anion exchange chromatography,membrane absorbers, reversed phase chromatography, normal phasechromatography, size exclusion chromatography, hydrophobic interactionchromatography, mixed mode chromatography, affinity chromatography,hydroxyapatite (HA) chromatography, or combinations thereof and morepreferably reversed phase chromatography; and C4) conditioning and/oroptionally purifying of the solution comprising the transcribed RNAobtained after step C3) by one or more steps of TFF.

In a preferred embodiment, the optional termination of transcription ofstep C1) may comprise the addition of an effective amount of a cationcomplexing agent such as EDTA. EDTA may efficiently stop the in vitrotranscription reaction and also deactivates nucleases and stabilizes theRNA due to the depletion of divalent cations. Moreover, addition of EDTAto the IVT-mix results in partial reduction of potentially occurringturbidity. It was also found that the addition of EDTA allows higherflow rates during subsequent TFF steps.

In a preferred embodiment, from about 10 to about 100 mM EDTA,preferably from about 10 to about 50 mM EDTA and even more preferablyfrom about 20 to about 30 mM EDTA is added. In a particularly preferredembodiment, 25 mM EDTA is added.

It was surprisingly found that TFF is particularly suitable for theconditioning of the solution comprising the transcribed RNA compared toother alternative conditioning methods such as using fast performanceliquid chromatography (FPLC) column affinity chromatography which showedirreversible binding of RNA and RNA elution could only be achieved bywashing the column with NaOH or most RNA was found in the flow-through.

In another preferred embodiment, step C4 comprises at least a firstdiafiltration step using TFF as described above; more preferably atleast a first diafiltration step using TFF and at least a seconddiafiltration step as described above; and even more preferably at leastone concentration step using TFF as described above, at least a firstdiafiltration step using TFF and at least a second diafiltration step asdescribed above.

Thus, in a preferred embodiment the at least one first step ofdiafiltration in step C4 is performed with an aqueous salt solution asdiafiltration solution. In a preferred embodiment, the salt of theaqueous salt solution may comprise alkaline metal halides such as NaCl,LiCl or KCl; organic salts such as NaOAc, earth alkaline metal halidessuch as CaCl₂); alkaline metal phosphates such as Na₃PO₄, Na₂HPO₄,NaH₂PO₄; or combinations thereof. In a preferred embodiment, the salt ofthe aqueous salt solution may comprise alkaline metal halides such asNaCl, LiCl or KCl; earth alkaline metal halides such as CaCl₂). In amore preferred embodiment, the aqueous salt solution comprises fromabout 0.1 M alkaline metal halide to about 1 M alkaline metal halide,more preferably from about 0.2 to about 0.5 M alkaline metal halide. Inanother preferred embodiment, the aqueous salt solution comprises NaCl.In a more preferred embodiment, the aqueous salt solution comprisesabout 0.1 M NaCl to about 1 M NaCl, more preferably from about 0.2 toabout 0.5 M NaCl. In a particularly preferred embodiment, the aqueoussalt solution comprises 0.2 M NaCl. In another preferred embodiment, theaqueous salt solution does not comprise buffering salts. The presence ofthe salt may be advantageous for removing contaminating spermidine fromthe RNA-Pool. In a preferred embodiment, the first diafiltration step isperformed using from about 1 to about 20 DV diafiltration solution,preferably from about 1 to about 15 DV diafiltration solution and morepreferably from about 5 to about 12 DV diafiltration solution and evenmore preferably from about 7 to about 10 DV diafiltration solution. In aparticularly preferred embodiment, the first diafiltration step isperformed using about 10 DV diafiltration solution.

In a preferred embodiment, the at least one second diafiltration step isperformed using water as diafiltration solution. In a preferredembodiment, the first diafiltration step is performed using from about 1to about 20 DV diafiltration solution, preferably from about 1 to about15 DV diafiltration solution and more preferably from about 5 to about12 DV diafiltration solution and even more preferably from about 6 toabout 10 DV diafiltration solution. In a particularly preferredembodiment, the second diafiltration step is performed using about 10 DVdiafiltration solution. In a preferred embodiment, a step ofconcentrating the RNA using TFF is performed after the seconddiafiltration step.

In a particularly preferred embodiment, the diafiltration solution doesnot comprise buffering salts in all TFF steps of the inventive method.

It is particularly preferred that all TFF steps according to the presentinvention, i.e. steps A3, C2 and C4 as defined herein, are performedwith the same type of TFF membrane, preferably with the same type of TFFmembrane cassette. Even more preferably, all TFF steps according to thepresent invention are performed with a TFF membrane cassette comprisingan mPES-based filter membrane with a MWCO of 100 kDa or acellulose-based filter membrane with a MWCO of 100 kDa, e.g.,commercially available TFF membrane cassettes such as NovaSep mPES witha MWCO of 100 kDa and Hydrosart (Sartorius) cellulose-based membranecassette with a MWCO of 100 kDa. Most preferably, all TFF stepsaccording to the present invention are performed with a TFF membranecassette comprising a cellulose-based filter membrane with a MWCO of 100kDa.

The skilled person will readily appreciate that the above comments onstep A3 also apply to steps d) and e) and to step iv). Similarly, theabove comments on step C2 also apply to step h) and the comments on stepC4 also apply to steps j) and k), respectively.

Optional Steps D to F:

In yet another embodiment, the method according to the present inventioncomprises at least one additional formulation step D), e.g. thecomplexation of the purified RNA with polycationic compounds, such aspolycationic polymers or polycationic peptides or proteins, e.g.protamine.

In this context the depletion of spermidine is absolutely necessary inorder to provide a sufficient complexation with polycationic compounds.

In yet another embodiment, the method according to the present inventionfurther comprises steps of E) filling and/or F) lyophilization.

The present invention was made with support from the Government underAgreement No. HRO011-11-3-0001 awarded by DARPA. The Government hascertain rights in the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B: Feed stream passes parallel to the membrane face as oneportion passes through the membrane (permeate/filtrate) (FIG. 1A) whilethe remainder (retentate) is recirculated from the filtration moduleback to the feed reservoir (FIG. 1B; adapted from Millipore literatureNo. TB032); concentration, diafiltration (desalting and bufferexchange), and fractionation of large from small molecules are possible.

FIGS. 2A-B: Results of MWCO screening with linearized pDNA FIG. 2A showsthe results of MWCO screening experiments from linearization reactionsof three different plasmids: P0625 (2626 bp), P1040 (3907 bp), and P0532(7362 bp). It can be easily seen that using spin filters of bothmanufacturers with 100 kDa MWCO retained the three different linearizedpDNAs almost completely, whereas higher MWCO lead to loss of linearizedplasmid DNA.

FIG. 2B shows DNA agarose gel electrophoresis of MWCO screening with thelinearized plasmid P1040 (3907 bp). The samples of linearization ofP1040 and the resulting filtration samples are shown in the DNA agarosegel electrophoresis. LA: 0.5 μg of the linearization reaction ascontrol. The analysed samples were diluted, and the fixed amount of 0.5μg DNA per lane was applied to the gel. If the DNA concentration was toolow, the maximum amount of DNA was applied. Due to the standardizationin DNA application, no quantitative statement can be given regarding theretaining of DNA. However the analysis shows that the integrity of DNAdid not decrease during filtration. Furthermore it can be seen from thegel that higher MWCO than 100 kDa lead to a loss of plasmid DNA in theretentate.

FIGS. 3A-B Results of MWCO screening with mRNA.

FIG. 3A shows the results of MWCO screening of RNA after transcriptionreaction for three different mRNA lengths (R1871: 589 nt, R1265: 1870nt, R 1626: 5337 nt). It can be easily seen that using spin filters ofboth manufacturer's with 100 kDa MWCO retain the three different mRNAscompletely.

FIG. 3B shows RNA agarose gel electrophoresis for the resultingfiltration samples of the RNA R1265. The analysed samples were diluted,and the fixed amount of 1 μg mRNA per lane was applied to the gel. Ifthe mRNA concentration was too low, the maximum amount of mRNA wasapplied. Due to that standardization in mRNA application, noquantitative statement can be given regarding the retaining of mRNA.However the analysis shows that the integrity of mRNA did not decreaseduring filtration. The gel reflected the same results, as measured inRNA concentration measurement, that the RNA R1265 was retainedcompletely by the 100 kDa membrane.

FIG. 4 : Flow rate screening of hollow fibre module. RNA in WFI (3775nt, mRNA after transcription reaction, diafiltrated in WFI) was used toperform a flow rate screening using a hollow fibre membrane (GE, PES,100 kDa, 50 cm²). As shown above, an increase in feed flow (FF) led toan increase in permeate flux, also the addition of retentate pressure(0.5 bar) showed an impact on permeate flux rate.

FIG. 5 : Permeate flux rates of different TFF cassettes at dp and TMP of1 bar.

The flux rates for three different TFF cassettes were in a range of 126to 140 l/h/m².

FIG. 6 : RNA stability after transcription reaction (A) and aftersubsequent TFF diafiltration to WFI for several hours (B). RNA integrity(relative area of full-length product) was determined by analyticalRP-HPLC after storage at different temperatures (room temperature, 5° C.and −20° C.). The in vitro transcription reaction without TFF wasanalyzed for RNA integrity after 1, 3, 5, 7, 10, 13, 14, 53 and 61 days.The in vitro transcription reaction after TFF was analyzed for RNAintegrity after 1, 5, 7, 12, 14 and 33 days.

FIG. 7 : Test of different membranes for spermidine depletion step Twomembranes (Novasep mPES 100 kDa and the cellulose-based SartoriusHydrosart 100 kDa) were tested for the spermidine depletion step using0.2 M NaCl for diafiltration. Both membranes showed comparable FLUXrates.

FIGS. 8A-B: Test of different membranes for TFF of linearizationreaction with higher membrane load

For concentration and diafiltration of the linearization reaction TFFmembranes made from different material, with a MWCO of 100 kDa andmembrane area of 200 cm² from different suppliers (The PES-basedmembranes Sartocon Slice 200 from Sartorius and the NovaSet-LS ProStream(Low Binding mPES) from NovaSep and the cellulose-based membraneSartocon Slice 200, Hydrosart from Sartorius) were tested with a highmembrane load (5.6 and 6 g plasmid DNA/m²).

Flux rates in the concentration step are shown in FIG. 8A. Thelinearization reaction was concentrated from 0.2 g/l to approximately1.5 g/l. The following parameters were used: dp and TMP=1 bar (P1=1.5bar, P2=0.5 bar and P3=0 bar) Flux rates in the diafiltration step areshown in FIG. 8B. The linearization reaction was diafiltrated against 10diafiltration volumes WFI with the same parameters used forconcentration.

All tested membranes showed similar results. During concentration of thelinearization reaction (FIG. 8A), FLUX rates decreased rapidly, butduring diafiltration in WFI (FIG. 8B) the FLUX-rates increased again.Both PES-based membranes (Sartorius PES and NovaSet mPES) showed similarresults, however, the Hydrosart membrane (Sartorius) showed higherpermeate flow rates.

FIG. 9 : Agarose gel electrophoresis of linearized pDNA

-   -   1.) DNA size marker    -   2.) TFF permeate after concentration (10 μl)    -   3.) TFF permeate after diafiltration (10 μl)    -   4.) TFF retentate (3 μl of 0.1 g/1)    -   5.) Control linearized plasmid (1.8 μl of 0.17 g/1)    -   6.) Control circular plasmid (3 μl of 0.1 g/1)    -   7.) Empty    -   8.) DNA size marker

Only a negligible amount of plasmid DNA is visible in the permeate ofthe concentration step and of the diafiltration step.

FIG. 10 : RNA agarose gel electrophoresis of permeate and retentatesamples taken during TFF of the RP-HPLC pools

-   -   1.) RNA marker    -   2.) RP-HPLC Pool I    -   3.) RP-HPLC Pool II    -   4.) RP-HPLC Pool III    -   5.) TFF permeate    -   6.) TFF permeate (40× concentrated)    -   7.) TFF retentate Pool I    -   8.) TFF retentate Pool II    -   9.) TFF retentate Pool III    -   10.) Final product    -   11.) Control    -   12.) Control    -   13.) empty    -   14.) RNA marker

FIG. 11 : Protein SDS PAGE samples taken during RNA production andpurification process

-   -   1 Protein Marker    -   2 TFF retentate after linearization    -   3 transcription reaction    -   4 TFF retentate before RP-HPLC    -   5 TFF retentate before RP-HPLC    -   6 TFF retentate before RP-HPLC    -   7 TFF retentate after RP-HPLC Pool I    -   8 TFF retentate after RP-HPLC Pool II    -   9 TFF retentate after RP-HPLC Pool III    -   10 Final Product    -   11 control    -   12 Protein Marker

FIG. 12 : TFF permeate flux rates. Concentration of pDNA linearizationreaction from 0.2 to 1.5 g/l DNA, using Hydrosart 100 kDa, dp and TMP 1bar; Membrane load: 6 g/m²; Concentration from 0.2 g/l DNA to 1.5 g/lDNA.

FIG. 13 : TFF permeate flux rates. Diafiltration of linearized pDNA into10 DFV WFI using Hydrosart 100 kDa, dp and TMP 1 bar; Membrane load: 6g/m².

FIG. 14 : TFF permeate flux rates. Diafiltration of RNA IVT-mix into 10DFV WFI using Hydrosart 100 kDa, dp and TMP 1 bar; Membrane load: 56g/m²;

FIG. 15 : TFF permeate flux rates. Concentration of RP-HPLC RNA pool,using Hydrosart 100 kDa, dp and TMP1 bar; Membrane load: 20 g/m²;Concentration from 0.1 g/l RNA to 5 g/l RNA; Temperature 17° C.

FIG. 16 : TFF permeate flux rates. Diafiltration of RNA, using Hydrosart100 kDa, dp and TMP 1 bar. (A) Diafiltration of RNA into 10 DFV 0.2 MNaCl; (B) Diafiltration of RNA into 10 DFV WFI.

EXAMPLES Example 1—Materials

The following materials of Table 1 were used in the subsequentexperimental section:

TABLE 1 Materials Equipment Manufacturer Vivaflow 50, PES, 100 kDaSartorius Sartocon Slice 200 100 kDa, PES Sartorius Sartocon Slice 200100 kDa, Hydrosart Sartorius (cellulose-based membrane) Sartcocon Slice200 300 kDa, PES Sartorius Omega Centramate T OS100T02, PES PALL GmbH100 kDa NovaSet-LS ProStream (Low Binding mPES), NovaSep 100 kDa Hollowfibre module, 100 kDa, PES GE-Healthcare Spin-Filter Nanosep ® (100kDa), PES Pall GmbH Spin-Filter Nanosep ® (300 kDa), PES Pall GmbHSpin-Filter Nanosep ® (1000 kDa), PES Pall GmbH Spin-Filter Vivaspin ®500 (100 kDa), PES Sartorius Spin-Filter Vivaspin ® 500 (300 kDa), PESSartorius Spin-Filter Vivaspin ® 500 (1000 kDa), PES Sartorius Vivaflow50 Modul, PES, 100 kDa Sartorius Sartoflow Slice 200, PES, 100 kDaSartorius

General Methods:

Example 2—Linearization of Plasmid DNA

The following conditions were used for linearization of the plasmid DNA:

1 μg plasmid DNA

0.5 μl reaction buffer

3 Units restriction enzyme EcoRI

Add. 5 μl with WFI (water for injection)

The reaction was incubated for 3 hours at 37° C. and stopped byheat-inactivation of the restriction enzyme (65° C., 30 minutes).

Example 3—General Description of the TFF Process

All tubes and the retentate vessel were cleaned with 75% EtOH and waterand were assembled.

The membrane cassette was fixed into the corresponding holder, accordingto the manufacturer's instruction, and respectively the hollow fibremembrane was connected to the system.

Afterwards, the system and membrane was flushed with at least 1 L water,1 L 1 M or 0.5 M NaOH for 1 hour (for removal of potential contaminants,like RNases) and was washed again with water, until pH value in thepermeate was neutral. Subsequently, the whole system was flushed withwater for injection (WFI) or diafiltration solution or buffer.

3.1—Concentration Step

DNA/RNA-solution was filled into the retentate vessel, and wasoptionally concentrated to the required concentration, by setting theindicated pressures.

3.2—Diafiltration Step

After the optional concentration step, diafiltration was started.Therefore the diafiltration tube was placed into the diafiltrationsolution or buffer. During diafiltration, the amount of permeate whichleft the system was automatically replaced by diafiltration solution orbuffer, due to the emerging vacuum. When the required diafiltrationvolume (dv) was reached, a different diafiltration solution or bufferwas optionally added to the system, and a second diafiltration step wascarried out. Before ending of the TFF step, the retentate was optionallyagain concentrated to the required volume, before withdrawal of theretentate from the system. Subsequently the system was flushed with 25mL WFI (water for injection) or buffer (permeate valve closed). Flushingliquid was optionally pooled with the TFF retentate. Optionally, RNA/DNAconcentration was measured and recovery of nucleic acid calculated.

3.3—System/Membrane Maintenance

After usage of the membranes, the cassette was flushed with 0.5 L water,subsequently with 0.5 M or 1 M NaOH for 1 hour, and again with water,until pH in the permeate was neutral. Afterwards, the water permeateflux value was determined to verify the cleanness of the membrane. Atthe end, the membrane was removed from the TFF system and stored ineither 0.1 M NaOH or in 20% EtOH. The TFF system was afterwards cleanedwith 75% EtOH and water and stored dryly.

Example 4—In Vitro Transcription

4.1—In vitro transcription

The linearized DNA plasmids were transcribed in vitro using T7polymerase. The in vitro transcription was performed in the presence ofa CAP analog (m7GpppG). The in vitro transcription was carried out in5.8 mM m7G(5′)ppp(5′)G Cap analog, 4 mM ATP, 4 mM CTP, 4 mM UTP, and1.45 mM GTP, 50 μg/ml DNA plasmid, 80 mM HEPES, 24 mM MgCl₂, 2 mMSpermidine, 40 mM DTT, 100 U/μg DNA T7 RNA polymerase, 5 U/μg DNApyrophosphatase, and 0.2 U/μl RNAse inhibitor.

The in vitro transcription reaction was incubated for 4 hours at 37° C.

After transcription the reaction was stopped by adding ETDA to a finalconcentration of 25 mM.

4.2—DNA Template Removal: DNase I Treatment

To digest DNA template 6 μl DNAse I (1 mg/ml) and 0.2 μl CaCl₂) solution(0.1 M)/μg plasmid DNA were added to the transcription reaction, andincubated for 3 h at 37° C.

Example 5—HPLC Purification of the RNA

The RNA was purified using PureMessenger® (CureVac, Tubingen, Germany;WO2008/077592A1).

Briefly, the TFF conditioned transcription reaction was purified usingReversed-Phase High pressure liquid chromatography (RP-HPLC). TheRP-HPLC was performed with a macroporous styrene/divinylbenzene column(particle size 30 μm, pore size 4000 Å) and column dimensions of 21.2mm×250 mm (volume 88.25 ml).

1 g/l RNA in 100 mM triethylammonium acetate (TEAA) was prepared,filtered with a 5 μm PVDF filter and used for preparative RP-HPLC. Aftermounting the column (stored in 88% acetonitrile), the storage solutionwas washed out with ultra-pure water. Next, the RNA sample was loadedonto the column and eluted with an eluent B/eluent A—gradient (eluent A:100 mM triethylammonium acetate (TEAA) in (water for injection (WFI), pH7.0; eluent B: 100 mM TEAA in 25% acetonitrile) starting with 100%eluent A, and ending with 100% eluent B.

During the elution procedure, fractions were automatically collected.Subsequently fractions were analyzed for RNA content by photometricaldetermination (A260) and for RNA integrity by agarose gelelectrophoresis or analytical HPLC.

Example 6—Analytical Methods

6.1—RNA Gel Electrophoresis

RNA was separated in formaldehyde-containing agarose gels (0.7% w/wformaldehyde, 1.2% w/v agarose) in 3-Morpholinopropane sulfonic acidbuffer (for details on method see Sambrook, Russel: Molecular Cloning: ALaboratory Manual, vol. 3, Cold Spring Harbor Laboratory, 2000.). RNAsamples were denatured in RNA sample buffer (Thermo scientific) at 80°C. for 5 min before loading on the gel. 1 μg of RNA was loaded per lane.

6.2—Protein Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed with ready-to-use 12% Mini-PROTEAN TGX gels(Bio-Rad). 4× Laemmli sample loading buffer and 10×SDS-PAGE runningbuffer were purchased from Bio-Rad. Samples were mixed with 4× loadingbuffer and incubated at 95° C. for 5 min. Sample load was normalized to10 μg RNA per lane. A voltage of 150 mV (corresponding to approx. 35 mAper gel) was applied until the smallest marker band reached the lowerend of the gel. Visualization of protein bands was performed withready-to-use Simply Blue Safe Stain (Invitrogen) according to theprotocol of the manufacturer. Alternatively, the Pierce Silver Stain Kit(Thermo Scientific) was used in order to increase staining sensitivity.

6.3—Quantification of RNA-Bound Spermidine

Spermidine was quantified by a modified protocol as described in Floreset al. (Plant Physiol. (1982) 69, 701-706)). Briefly, spermidine isbenzoylated under alkaline conditions followed by extraction withdiethylether. Benzoylated spermidine is detected and quantified by HPLCor mass spectrometry. Hexamethylene diamine is used as an internalstandard.

6.4—Determination of Residual Solvents

The content of residual solvents in the RNA sample was determined usingquantitative gas-chromatography with flame ionization detector (GC-FID).

Pilot Tests:

Example 7—Pore Screening Experiments

7.1—MWCO Screening with Plasmid DNA.

For determination of suitable MWCO (molecular weight cut-off) ofmembranes used in TFF, a MWCO-screening was conducted. Therefore spinfilters were used, as they require small volumes. Spin filters from twodifferent manufacturers (Spin-Filter Nanosep® (100 kDa, 300 kDa and 1000kDa), PES from PALL GmbH and Spin-Filter Vivaspin® 500 (100 kDa, 300 kDaand 1000 kDa), PES from Sartorius) were tested with a MWCO of 100, 300and 1000 kDa. Prior to use, the spin filters were flushed with 500 μlWFI (water for injection), and WFI was removed from the permeatecompartment completely. Subsequently, 300 μl of three differentlinearization reactions (see Example 2) were added and centrifuged atroom temperature, according to the manufacturer's instructions. Afterapproximately half the volume had passed the membrane, centrifugationwas stopped and the exact volume in the permeate and retentate chamberwas determined. Retentate chamber was then flushed with 100 μl WFI andcombined with the retentate. Beside volume measurement, also theconcentration of DNA was determined in the starting solution andretentate solution. DNA concentration was determined photometrically bymeasuring the absorption at 260 nm. FIG. 2A, shows the results of MWCOscreening from linearization reactions of three different plasmids:P0625 (2626 bp), P1040 (3907 bp), P0532 (7362 bp). It can be easilyseen, that using spin filters of both manufacturer's with 100 kDa MWCOretain the three different linearized pDNAs almost completely, whereashigher MWCO leads to loss of linearized plasmid DNA.

The samples of linearization P1040 and the resulting filtration samplesare shown in FIG. 2B by DNA agarose gel electrophoresis. The analyzedsamples were diluted, and the fixed amount of 0.5 μg DNA per lane wereapplied to the gel, if the DNA concentration was too low, the maximumamount of DNA was applied. Due to the standardization in DNAapplication, no quantitative statement can be given regarding theretaining of DNA. However the analysis shows that the integrity of DNAdid not decrease during filtration. Furthermore it can be seen from thegel that higher MWCO than 100 kDa leads to a loss of plasmid DNA in theretentate. Therefore a MWCO of 100 kDa was chosen for furtherexperiments regarding TFF of linearized plasmid DNA.

7.2—MWCO Screening with RNA

The experiment was conducted in the same way, as described for MWCOscreening with pDNA (Example 7.1). Prior to use, the spin filters wereflushed with 500 μl WFI, and WFI was removed from the permeatecompartment completely. After that, 300 μl of three differenttranscription reactions were added and centrifuged at room temperature,according to the manufacturer's instructions. After approximately halfthe volume had passed the membrane, centrifugation was stopped and theexact volume in the permeate and retentate chamber was determined.Retentate chamber was then flushed with 100 μl WFI and combined with theretentate. Beside volume measurement, also the concentration of RNA wasdetermined photometrically by measuring the absorption at 260 nm in thestarting solution and retentate solution. FIG. 3A shows the results ofMWCO screening of RNA after transcription reaction according to Example4 for three different mRNA lengths (R1871: 589 nt, R1265: 1870 nt, R1626: 5337 nt). It can be easily seen, that using spin filters of bothmanufacturer's with 100 kDa MWCO retain the three different mRNAscompletely.

Furthermore in FIG. 3B RNA agarose gel electrophoresis is shown for theresulting filtration samples of the RNA R1265. The analysed samples werediluted, and the fixed amount of 1 μg mRNA per lane was applied to thegel, if the mRNA concentration was too low, the maximum amount of mRNAwas applied. Due to that standardization in mRNA application, noquantitative statement can be given, regarding the retaining of mRNA.However the analysis shows that the integrity of mRNA did not decreaseduring filtration. The gel reflects the same results, as measured in RNAconcentration measurement that the RNA R1265 is retained completely bythe 100 kDa membrane, with a higher MWCO RNA is visible in the permeate.Therefore a MWCO of 100 kDa was selected for further experimentsregarding TFF of RNA solutions.

Example 8—Parameter Screening of Different Membranes

The in vitro transcription reaction R2587 (according to Example 4)already diafiltrated in WFI was used for parameter screening. Thedifferent membranes (Sartocon Slice 200 100 kDa, PES from Sartorius,Sartocon Slice 200 100 kDa, Hydrosart (cellulose-based membrane) fromSartorius and NovaSet-LS ProStream (Low Binding mPES), 100 kDa fromNovasep) were screened in respect to permeate flux versus TMP and dp,respectively. The sample load was 0.1 mg RNA/cm² membrane. The Hydrosartmembrane (cellulose-based membrane from Sartorius) showed the highestpermeate flow rate compared to the other membranes (polyethersulfone(PES)-based membranes from Sartorius and NovaSep) tested. As can be seenfrom the results as shown in Table 2 a pressure difference over themembrane (dp) of at least 0.5 bar and a transmembrane pressure (TMP) ofat least 0.75 bar are necessary to reach a FLUX rate of at least 100l/h/m².

TABLE 2 FLUX rates resulting from different parameters selected forscreening experiments Sartorius, NovaSet, PES, 100 mPES, 100 Hydrosart,kDa kDa 100 kDa p1 p2 p3 dp TMP FLUX FLUX FLUX [bar] [bar] [bar] [bar][bar] [l/h/m²] [l/h/m²] [l/h/m²] 0.5 0 0 0.5 0.25 45 54.6 72 1 0.5 0 0.50.75 133.8 141.6 171 1.5 1 0 0.5 1.25 164.4 172.8 235.8 1 0 0 1 0.5 87.685.2 135.9 1.5 0.5 0 1 1 180 178.8 233.1 2 1 0 1 1.5 238.8 215.4 307.81.5 0 0 1.5 0.75 138.6 120 198.9 2 0.5 0 1.5 1.25 228.6 247.2 298.8 2.51 0 1.5 1.75 280.8 308.4 381.6 2 0 0 2 1 185.4 181.2 270 2.5 0.5 0 2 1.5270 308.1 352.8

Based on these experiments the following parameters were chosen for TFFof the transcription reaction:

TABLE 3 Selected parameters for TFF of the transcription reaction FeedRetentate Permeate pressure pressure pressure (p1) (p2) (p3) dp TMP 1.5bar 0.5 bar 0 bar 1 bar 1 bar (= 0.15 MPa) (= 0.05 MPa) (= 0 MPa) (= 0.1MPa) (= 0.1 MPa)

Example 9—TFF with Higher Membrane Load

9.1—TFF of Transcription Reaction Using a Hollow Fibre Module

An RNA in vitro transcription reaction (R2587 with 3775 nt),diafiltrated in WFI was used to perform a flow rate screening in hollowfibre membrane (Hollow fibre module, 100 kDa, PES, 50 cm² from GEHealthcare). The membrane load was about 2.0 mg RNA/cm². As shown inFIG. 4 , an increase in feed flow (FF) lead to an increase in permeateflux, also the addition of retentate pressure (0.05 MPa) showed animpact on permeate flux rate. The flux rates were between 5 and 85l/h/m² (see FIG. 4 ).

9.2—TFF Using TFF Cassette Modules

An RNA in vitro transcription reaction (R2312 with 1885 nt),diafiltrated in WFI was used to perform a TFF using the following TFFparameters (Table 4):

TABLE 4 TFF parameters using TFF membrane cassette modules FeedRetentate Permeate pressure pressure pressure (p1) (p2) (p3) dp TMP 0.15MPa 0.05 MPa 0 MPa 0.1 MPa 0.1 MPa

The membrane load was about 4.5 mg RNA/cm². The Sartorius PES, 100 kDa,the NovaSet-LS ProStream (Low Binding mPES), 100 kDa from Novasep aswell as the Hydrosart Sartorius 100 kDa (cellulose-based membrane)cassettes were used as TFF modules, respectively. The obtained permeateflux rates were higher than for the hollow fibre membranes with valuesbetween about 125-140 l/h/m² as shown in FIG. 5 . Therefore, thediafiltration process is faster using TFF membrane cassettes due to thehigher FLUX rates compared to the FLUX rates using hollow fibremembranes.

Example 10—RNA Stability During TFF

An mRNA containing sample (R2564; 2083 nt) was diafiltrated with WFI forseveral hours at room temperature using the Sartorius PES, 100 kDamembrane cassette and the TFF parameters as described above in Example9.2. The RNA stability after transcription reaction was compared withthe stability of RNA after subsequent TFF. The stability, i.e. the RNAintegrity (relative area of full-length product) was determined byanalytical RP-HPLC.

The results are summarized in FIG. 6 .

The stability data showed that mRNA in transcription reaction mix wasonly stable if stored at −20° C. for up to 60 days (85-90%); at 5° C.integrity started decreasing slowly from the beginning (it sank from 85%to 61% in 61 days). A very rapid decrease in integrity was measured(decline from 81% to 51% over 14 days) if the mRNA was stored at roomtemperature. On the other hand, mRNA after TFF of the transcriptionreaction mix as described above was stable over 30 days if stored at−20° C. and at 5° C. If the mRNA was stored at room temperature it stillshowed high integrities for at least 7 days and then slowly decreaseddown to 80% integrity in 33 days. From this experiment it can beconcluded, that a higher degree of RNA stability was achieved, by TFF ofmRNA in WFI in comparison to the stability of RNA in the in vitrotranscription reaction without purification by TFF.

Example 11-TFF of RNA Containing RP-HPLC Pool

11.1—TFF Parameters for Diafiltration of RP-HPLC Pool

RP-HPLC purified RNA samples (as described in Example 5) in WFI, 0.1 MTEAA, 13% acetonitrile were used for the parameter screening.

We screened the different membranes (Hydrosart (cellulose-basedmembrane) from Sartorius, Omega Centramate T OS100T02, PES 100 kDa fromPALL, and PES-based membranes with a MWCO of 100 kDa and 300 kDa fromSartorius) in respect to TMP vs. permeate flux and at different RNAconcentrations (Table 5). The different membranes behaved similar duringTMP screening experiments. In general, the higher the dp and TMP thehigher the measured FLUX is (Table 5). At higher TMP values the processtends to be controlled by formation of the cake layer (maximum permeateflux reached, permeate flux independent of TMP).

TABLE 5 Results of the parameter screening Sartorius Sartorius. PALL.RNA PES 100 Hydrosart. PES 300 Centramate. conc p1 p2 p3 dp TMP kDa 100kDa kDa 100 kDa [μg/μl] [bar] [bar] [bar] [bar] [bar] [l/h/m²] [l/h/m²][l/h/m²] [l/h/m²]] 0.1 0.5 0 0 0.5 0.25 43.2 73.8 49.2 94.8 0.1 1 0.5 00.5 0.75 104.4 160.2 120 182.4 0.1 1.5 1 0 0.5 1.25 118.8 171 129.6 2040.1 1 0 0 1 0.5 46.8 126.9 123.6 127.8 0.1 1.5 0.5 0 1 1 106.8 200.7182.4 207.6 0.1 2 1 0 1 1.5 130.2 211.5 194.4 258 0.1 1.5 0 0 1.5 0.7568.4 178.2 144 154.8 0.1 2 0.5 0 1.5 1.25 118.8 225.9 204 242.4 0.1 2.51 0 1.5 1.75 151.2 248.4 219.6 304.8 0.1 2 0 0 2 1 86.4 205.2 169.2199.2 0.1 2.5 0.5 0 2 1.5 129.6 260.1 224.4 282 0.1 3 1 0 2 2 165.6 0 0Sartorius Sartorius. PALL. RNA- PES 100 Hydrosart. PES 300 Centramate.conc p1 p2 p3 dp TMP kDa 100 kDa kDa 100 kDa [μg/μl] [bar] [bar] [bar][bar] [bar] [l/h/m²] [l/h/m²] [l/h/m²] [l/h/m²]] 1 0.5 0 0 0.5 0.25 22.862.4 42 56.4 1 1 0.5 0 0.5 0.75 65.7 95.4 78 108 1 1.5 1 0 0.5 1.25 76.597.2 85.2 117.6 1 1 0 0 1 0.5 42.3 105.6 74.4 84 1 1.5 0.5 0 1 1 87.3138 106.8 133.2 1 2 1 0 1 1.5 103.5 135.6 112.8 142.8 1 1.5 0 0 1.5 0.7559.4 136.8 99.6 112.8 1 2 0.5 0 1.5 1.25 99.9 160.8 124.8 159.6 1 2.5 10 1.5 1.75 130.8 165.6 130.8 176.4 1 2 0 0 2 1 75.6 166.2 121.2 139.2 12.5 0.5 0 2 1.5 118.8 178.8 138 184.8 1 3 1 0 2 2 Sartorius Sartorius.PALL. RNA- PES 100 Hydrosart. PES 300 Centramate. conc p1 p2 p3 dp TMPkDa 100 kDa kDa 100 kDa [μg/μl] [bar] [bar] [bar] [bar] [bar] [l/h/m²][l/h/m²] [l/h/m²] [l/h/m²]] 1.5 0.5 0 0 0.5 0.25 22.8 57.6 38.4 48.6 1.51 0.5 0 0.5 0.75 69.6 85.5 67.2 95.4 1.5 1.5 1 0 0.5 1.25 74.4 90 69.6109.8 1.5 1 0 0 1 0.5 38.4 104.4 67.2 84.6 1.5 1.5 0.5 0 1 1 78 126 92.4135 1.5 2 1 0 1 1.5 98.4 118.8 93.6 165 1.5 1.5 0 0 1.5 0.75 46.8 127.888.8 117 1.5 2 0.5 0 1.5 1.25 93.6 136.8 114 168 1.5 2.5 1 0 1.5 1.75114 126 189 1.5 2 0 0 2 1 73.2 117 147 1.5 2.5 0.5 0 2 1.5 110.4 133.2180 1.5 3 1 0 2 2

Although higher dp and TMP values might lead to an increase in FLUXrates, the following parameters for large scale experiments wereselected (Table 6):

TABLE 6 selected parameters for TFF of RP-HPLC pool Feed RetentatePermeate pressure pressure pressure (p1) (p2) (p3) dp TMP 0.1-0.2 MPa0.05-0.1 MPa 0 MPa 0.05-0.1 MPa 0.075-0.15 MPa

The ranges for TMP and dp as shown in Table 6 were selected becauseunder these conditions the process is not completely cake layer driven.Although higher dp values (specifically higher μl values) lead tofurther increase of the FLUX rate, application in large scale processesare impeded due to restrictions in pump force. Moreover, lower shearforce (lower dp and TMP values) is preferred in respect to RNAstability.

11.2—Spermidine Depletion Via TFF

In first experiments, the concentrated RP-HPLC pool was diafiltratedwith water (WFI). In this case a relatively high residual spermidineconcentration in the final RNA solution was observed. To eliminate thespermidine an additional diafiltration step before final diafiltrationinto water was introduced. Different diafiltration solutions (Table 7)were screened. Approximately 5-10 mL of a RNA (R2564) containingsolution after RP-HPLC purification was diafiltrated with 100-200 mLdiafiltration solution, followed by diafiltration with 100-200 mL water.Here, single-use Vivaflow PES-based membrane cassettes (Sartorius) witha MWCO between 10-100 kDa were applied. Samples were analyzed after 10,20, 30 and 40 diafiltration exchange volumes, respectively. Finally, theretentate was concentrated to approx. 0.5 g/L and the amount ofspermidine was determined as described in Example 6.3. As a control, theRNA was not conditioned using TFF but precipitated by lithium chlorideprecipitation (see e.g. Sambrook et al., Molecular Cloning, a laboratorymanual, 2nd edition, Cold Spring Harbor Laboratory Press 1989.) and theamount of contaminating spermidine was determined as described inExample 6.3.

The results are summarized in Table 7.

TABLE 7 Spermidine concentration after TFF using different diafiltrationsolutions Diafiltration buffer Spermidine concentration composition (μgspermidine/mg RNA) water 100.81 20 mM sodium phosphate pH 6.2 53.31 0.5Msodium chloride 0.01 0.2M sodium chloride 0.08 Lithium chlorideprecipitation 0.04

Diafiltration of the RP-HPLC pool using TFF with pure water or 20 mMsodium phosphate did not efficiently remove RNA-bound spermidine.Application of high salt diafiltration solutions, e.g. NaCl basedsolutions, resulted in substantially complete depletion of RNA-boundspermidine. Furthermore, salts (e.g. TEAA), organic solvents (TEA, ACN)were efficiently removed.

Subsequent optimization experiments have demonstrated that the NaClconcentration could be reduced to at least 0.2 M in order to increasepermeate flow rates during diafiltration without effecting spermidinedepletion efficiency. Direct addition of NaCl to the concentratedRP-HPLC pool (final concentration ˜0.5 M or 0.2 M, respectively)resulted in faster spermidine depletion by TFF (less diafiltrationsolution is needed to reach the spermidine depletion). Application ofhigher concentrations of NaCl in the diafiltration solution are notadvisable since this might lead to RNA precipitation and, consequently,blocking of the TFF membrane. The spermidine depletion step can beperformed after RP-HPLC purification or directly after in vitrotranscription.

11.3.—Test of Different Membranes for Spermidine Depletion Step:

Two membranes (Novasep mPES 100 kDa and the cellulose-based SartoriusHydrosart 100 kDa) were further analyzed. We tested both membranes athigher sample load (approx. 2 mg RNA/cm² membrane) for diafiltration.For concentration and diafiltration the following parameters werechosen: dp=1 bar and TMP=1.5 bar, membrane load: 2.0 mg mRNA/cm²membrane. mRNA in WFI, 0.1 M TEAA, 13% acetonitrile and 0.2 M NaCl wasconcentrated from 0.1 g/l to 5 g/l and FLUX rates were determined.Diafiltration against 10 diafiltration volumes (dv) of 0.2 M NaClsolution and 10 dv of WFI was performed.

Results:

The overall time for concentration and diafiltration of 380 mg mRNA wasvery similar: NovaSet: 2.8 h, Hydrosart: 2.68 h. Respective FLUX ratesfor the diafiltration step are shown in FIG. 7 .

Example 12—TFF of Linearization Reaction with Higher Membrane Load

For concentration and diafiltration of the linearization reaction wetested TFF membranes made from different material, with a MWCO of 100kDa and membrane area of 200 cm² from different suppliers (The PES-basedmembranes Sartocon Slice 200 from Sartorius and the NovaSet-LS ProStream(Low Binding mPES) from NovaSep and the cellulose-based membraneSartocon Slice 200, Hydrosart from Sartorius) with a high membrane load(5.6 and 6 g plasmid DNA/m²). The parameters selected for TFF of thetranscription reaction were applied in this step as well, as they showedgood results before. Therefore dp and TMP=1 bar (P1=1.5 bar, P2=0.5 barand P3=0 bar) were selected to concentrate pDNA (P1452.8 120 mg) from0.2 g/l to approximately 1.5 g/l and afterwards for diafiltrationagainst 10 diafiltration volumes WFI.

Results:

All tested membranes showed similar results. During concentration of thelinearization reaction (FIG. 8A), FLUX rates decreased rapidly, butduring diafiltration in WFI (FIG. 8B) the FLUX-rates increased again.Both PES-based membranes (Sartorius PES and NovaSet mPES) showed similarresults, however, the Hydrosart membrane (Sartorius) showed higherpermeate flow rates.

Example 13—Complete Process

13.1—Linearization of pDNA Linearization of the plasmid DNA P1141 wasconducted according to Example 2.

13.2—Concentration and Diafiltration of Linearized pDNA Using TFF

For tangential flow filtration of the linearization reaction, aVivaflow50 filter cassette (PES membrane, MWCO 100 kDa, Sartorius) wasused. Before assembling the diafiltration setup, all product contactingcomponents (tubes, feed tank etc.) were thoroughly washed with ethanoland water. Next, the setup was washed with ultra-pure water. Then, thesetup was chemically sanitized with 500 mM NaOH solution. Subsequently,the setup was washed with WFI until a pH of 7 was measured in theretentate. Then, the feed tank was filled with 150 ml linearizationreaction according to Example 2.

In a first concentration step, approximately 100 ml of restrictionreaction was filtered, to obtain 50 ml retentate with a higher pDNAconcentration. Next, a vacuum was applied to the feed tank, and the feedtube was connected to a WFI bottle. Then, the diafiltration procedurewith 10 volumes (500 ml in total) WFI was performed. Subsequently, theretentate was concentrated as much as possible. After concentration, theretentate was collected in a sterile 50 ml reaction tube. The retentatewas analyzed on a DNA agarose gel (see FIG. 9 ). Retentate solutionswere stored at −20° C. For agarose gel electrophoresis the DNAconcentration was determined by measuring the absorption at 260 nm. Theindicated amounts of DNA were used for gel electrophoresis.

Results

Photometrical Determination of the DNA Concentration after Concentrationand Diafiltration:

The DNA concentration of the different DNA samples was determined bymeasuring the absorption at 260 nm:

TABLE 8 Plasmid concentration Volume for Concentration agarose gel [g/l][μl] Permeate from concentration step n/a 10 Permeate from diafiltrationstep n/a 10 Retentate from diafiltration step 0.10 3 Linearized plasmid0.17 1.8 Plasmid control 0.10 3

The dsDNA concentration in the retentate was photometrically determinedto be 1.05 g/l in a final volume of 22 ml. The diafiltration of thelinearized pDNA using TFF yielded 96.6% of input DNA.

Agarose Gel Electrophoresis:

Only a negligible amount of plasmid DNA is visible in the permeate ofthe concentration step and of the diafiltration step.

13.3—RNA In Vitro Transcription

800 ml of in vitro transcription mix as described in Example 4 wasincubated for 3 h at 37° C. Next, CaCl₂) and DNase I was added andsubsequently incubated for 2 h at 37° C.

13.4—Diafiltration of the Transcription Reaction

The TFF system Sartoflow 200 with two PES membranes (Sartorius, 200 cm²,100 kDa) was used to exchange the buffer of three aliquots (400 ml each)of the transcription reaction to WFI (process parameters indicated inTable 9). First, container and tubes were cleaned with ultrapure water,ethanol and WFI. Then the setup was assembled and the filter cassettewas mounted on the Sartocon holder according to the manufacturer'sinstructions. The whole system was cleaned with ultra-pure water.Subsequently, the system was chemically sanitized by a 1 hour wash with1 M NaOH. Then, the setup was washed with WFI until a pH of 7 wasmeasured in the retentate and the system was equilibrated with 500 mlWFI. After that, 400 ml transcription reaction (see 13.3) were added tothe retentate reservoir, pressures (dp and TMP) were set to 1 bar, andthe diafiltration procedure was started against 10 diafiltration volumes(DFV) water for injection (WFI). After diafiltration by TFF the RNAconcentration was measured photometrically.

TABLE 9 TFF parameters used for diafiltration of the transcriptionreaction Parameters Amount of RNA [mg] 2100-2200 Volume of RNA-solution[ml] 410-430 membrane Sartorius, PES, 100 kDa Number of membranes 2pressures [bar] P1 = 1.5 P2 = 0.5 P3 = 0   TMP und dp = 1 barDiafiltration volume WFI [1] 4.1-4.3 Membrane load [mg RNA/cm²]   5-5.6

Results:

The final concentration of the RNA after diafiltration was 5 g/l and thetotal recovery rate of the RNA after buffer exchange via diafiltrationwas 98%.

Example 13.5—RP-HPLC Purification of the Conditioned TranscriptionReaction

The RNA solution obtained from Example 13.4 was diluted to 100 mM TEAAand a concentration of 1 g/l by addition of 1 M triethylammonium acetate(TEAA) and WFI. The RNA was step-wise purified according to Example 5.The HPLC fractions were collected, the product-containing fractions werepooled and divided in three pools I to III.

Moreover, fractions were analyzed for RNA content (UV260/280) and everyfraction was analyzed for RNA integrity.

Results:

TABLE 10 Determination of RNA concentration and RNA integrity afterRP-HPLC purification RNA RNA Pools Volume conc. amount integrity Pool ICa. 7.2 L 0.12 g/l Ca. 840 mg 97.8% Pool II Ca. 7.3 L 0.12 g/l Ca. 840mg 97.7% Pool III Ca. 6.2 L 0.12 g/l Ca. 744 mg 95.7%

Compared to the starting material (TFF conditioned RNA), the integritycould be increased by >10% (86.5% RNA integrity before RP-HPLC) byseparating aborted RNA species.

Example 13.6—Concentration and Diafiltration of the RP-HPLC Pool Via TFF

The three RP-HPLC purified RNA pools were processed separately using anadditional concentration and diafiltration step with TFF to furtherseparate the RNA from impurities (e.g. spermidine contaminations) and toexchange the solvent.

First, every pool was concentrated from 0.12 g/l to approximately 5 g/lusing TFF. 5M NaCl solution was added to the retentate to get a finalconcentration of 0.2 M NaCl. Then, a diafiltration was performed against0.2 M NaCl (10 DFV) to remove spermidine impurities. Next, thediafiltration solution was exchanged to WFI (10 DFV). The processparameters are shown in Table 11. After TFF RNA concentration wasdetermined by measuring the absorption at 260 nm. For agarose gelelectrophoresis the indicated amounts of RNA were used for gelelectrophoresis.

TABLE 11 Parameters used for TFF of the RP-HPLC pools Pool 1 Pool 2 Pool3 Pressures P1 = 2   P1 = 2 P1 = 2 used for P2 = 0.5 P2 = 1 P2 = 1concentration P3 = 0   P3 = 0 P3 = 0 [bar] TMP = 1.2 TMP = 1.5 TMP = 1.5dp = 1.5 dp = 1.0 dp = 1.0 Pressures P1 = 2   P1 = 2 P1 = 2 used for P2= 1   P2 = 1 P2 = 1 diafiltration P3 = 0   P3 = 0 P3 = 0 [bar] TMP = 1.5TMP = 1.5 TMP = 1.5 dp = 1.0 dp = 1.0 dp = 1.0 TFF Hydrosart- Hydrosart-Hydrosart- membrane Membrane Membrane Membrane 200 cm²; 100 kDa 200 cm²;100 kDa 200 cm²; 100 kDa Membrane 2.1 mg RNA/cm² 2.1 mg RNA/cm² 1.9 mgRNA/cm² load start volume 7.2 L 7.3 L 6.2 L

Results:

The yield after the diafiltration was spectrometrically determined to be94.44%, 94.3% and 90.5% respectively.

Agarose Gel Electrophoresis:

Samples of the TFF diafiltration procedure after RP-HPLC purificationwere analyzed. Respective samples were analyzed using agarose gelelectrophoresis (see FIG. 10 ). Loading scheme of the respective RNAagarose gel is shown in table 12.

TABLE 12 Loading scheme of permeate and retentate samples taken duringTFF of the RP-HPLC pools. RNA conc. Volume used for agarose Lane Sample[μg/μl] gel electrophoresis [μl] 1 RNA marker 8 2 RP-HPLC 0.12 9 Pool I3 RP-HPLC 0.12 9 Pool II 4 RP-HPLC 0.12 9 Pool III 5 TFF permeate n.a. 96 TFF permeate n.a. 9 (40× concentrated) 7 TFF retentate 5.00 1 Pool I 8TFF retentate 5.00 1 Pool II 9 TFF retentate 4.84 1 Pool III 10 Finalproduct 4.84 1 11 Control 5.00 1 12 Control 1.00 1 13 empty 14 RNAmarker 8

The TFF permeate samples did not contain detectable RNA levels (eventhough samples that had been concentrated 40×).

The TFF retentate samples and the final TFF conditioned RNA poolcontained RNA of integrity of about 100%, all of those with a band sizeof 2476 bases, which was in accordance to the theoretically expectedsize.

Example 13.7—Determination of Protein Content Using a BCA Assay

To determine the protein content in the samples, the BCA-test was used.The total protein concentration contained in a sample was measuredphotometrically via absorption at 562 nm compared to a protein standard(bovine serum albumin, BSA). The test was performed using a commerciallyavailable BCA kit, according to the manufacturer's instructions.

To produce a 20 μg/ml bovine serum albumin (BSA) solution stocksolution, 20 μl BSA solution [1 mg/ml] was mixed with 980 μl water forinjection. This BSA stock solution was used to generate a standard curveusing BSA solutions of different concentrations (50 μl each, diluted inWFI): 2.5 μg/ml; 5 μg/ml; 10 μg/ml; 15 μg/ml; 20 μg/ml

The protein content was determined in samples from the TFF afterlinearization reaction (Example 13.2), from the transcription reaction(Example 13.3), before RP-HPLC (Example 13.4) and after RP-HPLC and TFFagainst 0.2 M NaCl (Examples 13.5 and 13.6).

Results:

The measurements were performed in a standard photometer. Results aredisplayed in Table 13.

TABLE 13 Determined protein concentrations A562 RNA/DNA nm (AU) proteinconc. protein/RNA Sample average Dilution [μg/ml] [mg/ml] [μg/mg] 1 TFFretentate after 0.717 20 324.2 1.05 308.8 linearization 2 transcriptionreaction 0.404 1000 6823.4 5.07 1345.8 3 TFF retentate before RP- 0.59610 122.9 4.30 28.6 HPLC 4 TFF retentate before RP- 0.632 10 136 5,.226.7 HPLC 5 TFF retentate before RP- 0.625 10 131.2 5.22 25.1 HPLC 6 TFFretentate after RP- 0.397 2 13.2 5.03 2.6 HPLC Pool I 7 TFF retentateafter RP- 0.352 2 10.7 5.00 2.1 HPLC Pool II 8 TFF retentate after RP-0.455 2 16.6 4.84 3.4 HPLC Pool III

Values of the BCA-assay are shown. 1: TFF retentate of linearizationreaction; 2: Transcription reaction; 3-5: TFF retentates oftranscription reactions before RP-HPLC; 6-8: TFF retentates oftranscription reactions after RP-HPLC

A step-wise depletion of the protein content per RNA over the whole RNApurification procedure could be observed.

SDS-PAGE:

SDS-PAGE was used to determine the protein content in the differentsamples. The indicated amounts of samples were used for SDS-PAGE. Theresults are shown in FIG. 11 .

TABLE 14 Samples used for SDS PAGE RNA/DNA Volume Lane Sample conc.[mg/ml] [μl] 1 Protein Marker 2 TFF retentate after 1.05 9.5linearization 3 transcription reaction 5.07 2.0 4 TFF retentate before4.30 2.3 RP-HPLC 5 TFF retentate before 5,.2 2.0 RP-HPLC 6 TFF retentatebefore 5.22 1.9 RP-HPLC 7 TFF retentate after 5.03 2.0 RP-HPLC Pool I 8TFF retentate after 5.00 2.0 RP-HPLC Pool II 9 TFF retentate after 4.842.1 RP-HPLC Pool III 10 Final Product 4.84 2.1 11 control 5.00 2.0 12Protein Marker

No protein bands were detectable in samples after RP-HPLC purification.

Example 13.8—Determination of Spermidine Concentration

Spermidine concentration was measured in the TFF retentates beforeRP-HPLC (Example 13.4) and after RP-HPLC purification and TFF against0.2 M NaCl (Example 13.5 and 13.6) according to Example 6.3.

Results:

TABLE 15 Spermidine concentrations in RNA samples RNA conc.Spermidin/RNA Sample [g/l] [ng/mg] TFF retentate before 0.22 39964.50RP-HPLC (1:20) TFF retentate before 0.26 39039.21 RP-HPLC (1:20) TFFretentate before 0.26 37748.26 RP-HPLC (1:20) RP-HPLC-Pool I 0.121834.98 RP-HPLC-Pool II 0.12 30.08 RP-HPLC-Pool III 0.12 110.90 TFFretentate after 5.03 1.00 RP-Pool I TFF retentate after 5.00 3.08RP-Pool II TFF retentate after 4.84 1.48 RP-Pool III

Results:

Spermidine was detectable in samples of the TFF retentates beforeRP-HPLC purification. In the samples purified by RP-HPLC and TFF using0.2 M NaCl only a very low amount of spermidine was detectable (seeTable 15).

Example 13.9—Determination of Organic Solvents

The concentration of acetonitrile (ACN) and TEAA was determinedaccording to Example 6.4.

Results:

The final sample after RP-HPLC purification and TFF contained less than40 ppm ACN and less than 2 ppm TEAA.

Example 14—Overview of the Process and Key Process Parameters

In the following, a further example of the inventive method isillustrated, providing process parameters of the method for each of theindividual steps including concentration of the linearization reactionand diafiltration of linearized plasmid DNA, diafiltration of RNA invitro transcription reaction and concentration and diafiltration of anRP-HPLC RNA pool.

14.1—Concentration of the Linearization Reaction and Diafiltration ofLinearized Plasmid DNA Using TFF:

Linearization of the plasmid DNA was performed as described in Example2. For tangential flow filtration of the linearization reaction(conducted according to Example 2), a Hydrosart filter cassette(cellulose based membrane, MWCO 100 kDa, Sartorius) was used. Theplasmid DNA concentration procedure as well as the diafiltrationprocedure was performed as explained above (see Example 13). The resultof the concentration of the linearization mix is provided in FIG. 12 .The result of the diafiltration of the linearized plasmid DNA isprovided in FIG. 13 . Relevant process parameters are summarized inTable 16.

TABLE 16 TFF process parameters of plasmid DNA concentration anddiafiltration Process parameter Concentration Initial pDNA concentration0.2 of the [g/l] linearization Pressures used for concentration P1 = 1.5reaction of the pDNA linearization P2 = 0.5 mix [bar] P3 = 0   TMP = 1dp = 1 Obtained pDNA concentration 1.0 or 1.5 [g/l] Diafiltration ofPressures used for diafiltration P1 = 1.5 the linearized of linearizedpDNA [bar] P2 = 0.5 pDNA in WFI P3 = 0   TMP = 1 dp = 1 DFV 10 DF bufferWFI General TFF membrane cassettes Hydrosart parameters membranecassette; 200 cm²; 100 kDa or NovaSet-LS ProStream (Low Binding mPES),100 kDa Membrane load 0.1-0.6 mg DNA/cm² Feed flowrate [l/h/m²] 750-900Permeate flux rate [l/h/m²]  30-100

14.2—Diafiltration of the RNA IVT Reaction by TFF:

RNA in vitro transcription was performed as described in Example 4. Fortangential flow filtration of the RNA IVT reaction (conducted accordingto Example 4), a Hydrosart filter cassette (cellulose based membrane,MWCO 100 kDa, Sartorius) was used. The conditioning of the RNA IVTreaction was performed as explained above (see Example 13). The resultof the diafiltration of the RNA IVT reaction is provided in FIG. 14 .Relevant process parameters are summarized in Table 17.

TABLE 17 TFF Process parameters of the RNA IVT reaction diafiltrationProcess parameter Pressures used for diafiltration of P1 = 1.5 RNA IVTreaction [bar] P2 = 0.5 P3 = 0   TMP = 1-1.5 dp = 1 DFV 10 DF buffer WFITFF membrane cassettes Hydrosart membrane cassette; 200 cm²; 100 kDa orNovaSet-LS ProStream (Low Binding mPES), 100 kDa Membrane load 2.5-6.5mg RNA/cm² Feed flowrate [l/h/m²] 300-1050 Permeate flux rate [l/h/m²]20-120

14.3—Concentration and Diafiltration of the RP-HPLC RNA Pool

The in vitro transcribed RNA was purified by RP-HPLC as described inExample 5. For tangential flow filtration of the RP-HPLC RNA pool, aHydrosart filter cassette (cellulose based membrane, MWCO 100 kDa,Sartorius) was used. The concentration of the RP-HPLC RNA pool wasperformed as explained above (see Example 13). The result of theconcentration of the RP-HPLC RNA is provided in FIG. 15 . Thediafiltration of the RNA into 0.2 M NaCl and a further diafiltration ofthe RNA into WFI was performed as explained above (see Example 13). Theresult of the diafiltration of the RP-HPLC RNA pool is provided in FIG.16 . Relevant process parameters are summarized in Table 18.

TABLE 18 TFF process parameters of the RP-HPLC RNA concentration anddiafiltration Process parameter Concentration Initial RNA concentration0.1 of the [g/l] RP-HPLC RNA Pressures used for P1 = 1.5 poolconcentration of the pDNA P2 = 0.5 linearization mix [bar] P3 = 0   TMP= 1 dp = 1 Obtained RNA 5 +/− 0.25 concentration [g/l] Diafiltration ofPressures used for P1 = 1.5 the RP-HPLC diafiltration of RNA P2 = 0.5RNA [bar] P3 = 0   pool in NaCl TMP = 1-1.5 buffer dp = 1 DFV 10 DFbuffer 0.2M NaCL Diafiltration of Pressures used for P1 = 1.5 theRP-HPLC diafiltration of RNA P2 = 0.5 RNA [bar] P3 = 0   pool in WFI TMP= 1-1.5 dp = 1 DFV 10 DF buffer WFI General TFF membrane cassettesHydrosart membrane parameters cassette; 200 cm²; 100 kDa or NovaSet-LSProStream (Low Binding mPES), 100 kDa Membrane load 2 mg-2.5 mg RNA/cm²Feed flowrate [l/h/m²] 900-1500 Permeate flux rate [l/h/m²] 25-140Temperature [° C.] 17° C. or <17° C.

Embodiment List:

1. A method for producing and purifying RNA, comprising the steps of

A) providing DNA encoding the RNA;

B) transcription of the DNA to yield a solution comprising transcribedRNA; and

C) conditioning and/or purifying of the solution comprising transcribedRNA by one or more steps of tangential flow filtration (TFF).

2. The method according to item 1, wherein in step A) plasmid DNA isprovided as DNA encoding the RNA and the method comprises subsequentlyto step A) the steps:

A1) linearization of the plasmid DNA in a linearization reaction;

A2) optionally termination of the linearization reaction; and

A3) conditioning and/or purifying of the linearization reactioncomprising linearized plasmid DNA by one or more steps of TFF.

3. The method according to item 1 or 2, wherein step C) comprises atleast one diafiltration step and/or at least one concentration stepusing TFF.

4. The method according to item 3, wherein the at least onediafiltration step using TFF in step C) comprises diafiltration with anaqueous salt solution.

5. The method according to item 4, wherein the aqueous salt solution isa NaCl solution, preferably an aqueous solution comprising from about0.1 M NaCl to about 1 M NaCl, more preferably a solution comprising fromabout 0.2 to about 0.5 M NaCl.

6. The method according to item 3, wherein the at least onediafiltration step using TFF of step C) comprises diafiltration withwater.

7. The method according to any one of items 1 to 6, wherein the methoddoes not comprise a step of phenol/chloroform extraction and/or DNAand/or RNA precipitation.

8. The method according to any one of items 1 to 7, wherein the methoddoes not comprise a step of using a TFF hollow fiber membrane.

9. The method according to any one of items 1 to 8, wherein the at leastone or more steps of TFF comprises using a TFF membrane with a molecularweight cutoff of 500 kDa, preferably of ≤200 kDa and most preferably of100 kDa.

10. The method according to any one of items 1 to 9, wherein the atleast one or more steps of TFF comprises using a TFF membrane comprisingat least one of polyethersulfone (PES), modified polyethersulfone(mPES), a cellulose derivative membrane or combinations thereof.

11. The method according to any one of items 1 to 10, wherein the atleast one or more steps of TFF comprises using a TFF membrane comprisinga cellulose derivative membrane with a molecular weight cutoff of about100 kDa.

12. The method according to any one of items 1 to 11, wherein the atleast one or more steps of TFF comprises using a TFF membrane cassette.

13. The method according to any one of items 1 to 12, wherein the methodcomprises in step C) at least one further purification method before orafter the one or more steps of TFF.

14. The method according to any of items 1 to 13, wherein the methodcomprises in step C) the steps:

C1) optionally termination of transcription;

C2) conditioning and/or purifying of the solution comprising thetranscribed RNA by one or more steps of TFF;

C3) purifying the RNA by any further purification method; and

C4) conditioning and/or optionally purifying of the solution comprisingthe transcribed RNA obtained after step C3) by one or more steps of TFF.

15. The method according to item 14, wherein step C2) comprises at leastone step of diafiltration using TFF with water and/or diafiltration withan aqueous salt solution, preferably an aqueous NaCl solution and morepreferably with an aqueous solution comprising from about 0.1 M NaCl toabout 1 M NaCl, more preferably a solution comprising from about 0.2 toabout 0.5 M NaCl.

16. The method according to item 14 or 15, wherein step C4) comprises atleast one first diafiltration step using TFF.

17. The method according to item 16, wherein step C4) comprises at leastone second diafiltration step using TFF.

18. The method according to any one of items 16 or 17, wherein the atleast one first diafiltration step using TFF in step C4) comprisesdiafiltration with an aqueous salt solution, preferably an aqueous NaClsolution and more preferably with an aqueous solution comprising fromabout 0.1 M NaCl to about 1 M NaCl, more preferably a solutioncomprising from about 0.2 to about 0.5 M NaCl.

19. The method according to item 17 or 18, wherein the seconddiafiltration step using TFF of step C4) comprises diafiltration withwater.

20. The method according to any one of items 13 to 19, wherein the atleast one further purification method is performed by means of highperformance liquid chromatography (HPLC) or low normal pressure liquidchromatography methods.

21. The method according to any one of items 13 to 20, wherein the atleast one further purification method is a reversed phase chromatographymethod.

22. The method according to any one of items 1 to 21, wherein all stepsof TFF are performed with the same TFF membrane.

23. The method according to any one of items 1 to 22, wherein thetranscribed RNA is selected from the group consisting of mRNA, viralRNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA),antisense RNA, CRISPR RNA, ribozymes, aptamers, riboswitches,immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), smallnuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), andPiwi-interacting RNA (piRNA) or whole-cell RNA and preferably is mRNA.

24. The method according to any one of items 1 to 23, wherein thetranscription of DNA in step B) is performed as in vitro transcription.

The invention claimed is:
 1. A method for producing purified RNA,comprising the steps of: A1) providing plasmid DNA encoding a RNA of 500to 10000 nucleotides in length; A2) linearizing the DNA with arestriction endonuclease to produce linearized DNA; B) transcribing thelinearized DNA to yield transcribed RNA, wherein said transcribing is ina solution comprising: nucleoside triphosphates (NTPs); T7 polymerase,spermidine, salts and a HEPES or TRIS buffer; and C) purifying thetranscribed RNA by performing at least one step of tangential flowfiltration (TFF) using a TFF membrane cassette, thereby producingpurified RNA, wherein the method comprises at least one step of DNA orRNA purification using chromatography.
 2. The method of claim 1, whereinthe at least one step of DNA or RNA purification using chromatographycomprises using anion exchange chromatography.
 3. The method of claim 1,wherein at least one step of TFF is performed after the chromatography.4. The method of claim 1, wherein C) purifying the transcribed RNAcomprises at least two steps of TFF.
 5. The method of claim 4, whereinthe at least two steps of TFF are both using a TFF membrane cassette. 6.The method of claim 1, wherein the TFF membrane cassette comprises acellulose-based TFF membrane.
 7. The method of claim 6, wherein C)purifying the transcribed RNA comprises performing at least one step ofTFF with an aqueous salt solution.
 8. The method of claim 7, wherein theaqueous salt solution is a NaCl solution or an organic salt solution. 9.The method of claim 7, wherein performing at least one step of TFFcomprises using a TFF membrane with a molecular weight cutoff of ≤500kDa.
 10. The method of claim 1, further comprising treating the solutionwith DNAse after said transcribing.
 11. The method of claim 1, whereinthe RNA is a mRNA.
 12. The method of claim 9, wherein the transcribingis in a buffer comprising 0.1 mM to 10 mM spermidine.
 13. The method ofclaim 12, wherein the method produces purified RNA with a reduced levelof spermidine relative to the level of spermidine in step B.
 14. Themethod of claim 11, wherein the mRNA comprises a modified nucleotide.15. The method of claim 14, wherein the modified nucleotide is1-methyl-pseudouridine.
 16. The method of claim 13, wherein thetranscribing is in a buffer comprising a HEPES buffer.
 17. The method ofclaim 13, wherein performing at least one step of TFF comprises using aTFF membrane with a molecular weight cutoff of about 100 kDa to 300 kDa.18. The method of claim 13, wherein the membrane cassette of step C2)has a MWCO of about 300 kDa.
 19. The method of claim 13, wherein the RNAis 500 to 5000 nucleotides in length.
 20. The method of claim 19,wherein the transcribing is in a buffer comprising a cap analog toproduce a capped RNA.
 21. The method of claim 20, wherein the capped RNAis a CAP1 capped RNA.
 22. The method of claim 21, wherein therestriction endonuclease is a type II restriction endonuclease.
 23. Themethod of claim 17, wherein the method further comprises formulating thepurified RNA.
 24. The method of claim 22, further comprising a fillingstep.
 25. The method of claim 17, wherein the TFF membrane cassettecomprises a stabilized cellulose-based TFF membrane.
 26. The method ofclaim 23, wherein formulating the purified RNA comprises complexing theRNA with a cationic compound.